HSF-like transcription factor, TBF1, is a major molecular switch for growth-to-defense transition in plants

ABSTRACT

The present invention relates to new methods to study and control the expression of plant genes, particularly nucleotide sequences located downstream from regions comprising binding sites for transcription factors, such as the cis-element translocon 1 (TL1) comprising GAAGAAGAA and similar sequences. The invention relates to isolated nucleotide sequences comprising a regulatory region comprising a promoter operably-linked to one or more upstream open reading frames (uORFs) and one or more downstream open reading frames (dORFs) encoding one or more functional polypeptides, including transcription factors such as TBF1, reporter polypeptides, and polypeptides conferring resistance to drugs, resistance of plants viral, bacterial, or fungal pathogens, and polypeptides involved in the growth of plants. Related aspects include the use of a region which encodes one or more polypeptides designated uORF1 and uORF2 from  Arabidopsis  plants, natural and synthetic variants of these polypeptides, and their homologues and orthologues isolated from other plant species, including crop plants, plus vectors, cells, plant propagation material, transgenic plants, and seeds comprising nucleic acids comprising said all or portions of said regulatory region. Other aspects relate to methods of using these regulatory elements to generate and screen for transgenic plants having improved resistance microbial and viral plant pathogens, and engineered cells and plants comprising these one or more of these genetic elements to facilitate the production of proteins for use in structure/function studies, in industrial, medical, and agricultural applications, particularly in the discovery of metabolic pathways involved in the and development of disease-resistant plants.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/310,320, filed Jun. 20, 2014, which is a continuation-in-part of PCT/US2012/070838, filed Dec. 20, 2012, published as WO 2013/096567 A2 on Jun. 27, 2013, claiming benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 61/578,632, filed Dec. 21, 2011.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with U.S. Government Support under Federal Grant Numbers MCB-0519898 and IOS-0929226, both awarded by the National Science Foundation, to X. Dong. The U.S. Government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF A SEQUENCE LISTING

The sequence listing contained in the file “761_183_008_US_ST25.txt”, created on Dec. 18, 2014, modified on Dec. 18, 2014, file size 235,438 bytes, is incorporated by reference in its entirety herein. The sequence listing contained in the file “127183_0007_US_ST25.txt”, created on Jun. 20, 2014, modified on Jun. 20, 2014, file size 235,395 bytes, is also incorporated by reference in its entirety herein. The originally-filed and amended sequence listings, if any, of PCT/US2012/070838, filed Dec. 20, 2012, U.S. Ser. No. 61/578,632, filed Dec. 21, 2011, are also incorporated by reference in their entireties.

INCORPORATION-BY-REFERENCE IN NON-PROVISIONAL APPLICATION UNDER 37 CFR 1.58 TO LARGE TABLES, INCLUDING SUPPLEMENTAL TABLES OF INFORMATION INCLUDED IN EARLIER PRIORITY APPLICATIONS

Table S1, which collectively refers to eight Supplemental Tables S1A-S1H, in the subsection labeled “Statistical Analysis” of the “Detailed Description of the Invention”, provides a summary of the complete lists of TBF1-dependent SA- and elf18-regulated genes set forth in tables formatted in Microsoft Word, extracted from eight worksheets of an Excel file.

The data in the eight Supplemental Tables S1A-S1H, which would occupy more than 580 pages if submitted on paper, are incorporated by reference in their entirety, herein, under 37 CFR 1.58. The data in these Supplemental Tables are contained in the following file: “DULV_D946US_Table_S1A-S1H_Supplemental_Tables.pdf”, modified on Dec. 21, 2011, file size 3,473,862 bytes, which was co-filed with and incorporated by reference in U.S. Provisional Application No. 61/578,632, filed Dec. 21, 2011. These tables were also incorporated by reference in the international application as PCT/US12/078038, filed Dec. 20, 2012, under Rule 20.6 to Supplemental Tables of Information Included In Earlier Priority Applications, and in Non-Provisional U.S. application Ser. No 14/310,320, filed Jun. 20, 2014.

FIELD OF THE INVENTION

The present invention relates to new methods to study and control the expression of plant genes, particularly genes located downstream from regions comprising binding sites for transcription factors, such as the cis-element translocon 1 (TL1) comprising GAAGAAGAA (SEQ ID NO: 99) and similar sequences. The invention relates to isolated nucleotide sequences comprising a regulatory region comprising a promoter operably-linked to one or more upstream open reading frames (uORFs) and one or more downstream open reading frames (dORFs) encoding one or more functional polypeptides, including transcription factors such as TBF1, reporter polypeptides, and polypeptides conferring resistance to drugs, resistance of plants viral, bacterial, or fungal pathogens, and polypeptides involved in the growth of plants. Another aspect of the invention relates to the use of a translational regulatory region wherein said uORFs encode polypeptides designated uORF1 and uORF2 from Arabidopsis plants, natural and synthetic variants of these polypeptides, and their homologues and orthologues isolated from other plant species, including crop plants. This regulatory region allows translation of dORFs in response to pathogen challenge. The invention is also directed to vectors, cells, plant propagation material, transgenic plants, and seeds comprising nucleic acids comprising said regulatory region. Other aspects relate to methods of using these regulatory elements to generate and screen for transgenic plants having improved resistance to disease, particularly microbial and viral plant pathogens. The invention is also directed to plants comprising said ORFs to facilitate the controlled production of one or more recombinant proteins in plant-based expression systems. Measurement of the amount or activity of a recombinant protein in this system can reflect the actions of one or more factors involved in the transcriptional and/or translational control signals, including promoters and uORFS upstream from the coding sequence for a polypeptide. The invention is also directed to engineered cells and plants comprising these genetic elements to facilitate the production of proteins for use in structure/function studies, in industrial, agricultural, and medical applications, and particularly in the understanding and development of disease-resistant plants.

BACKGROUND OF THE INVENTION

The sessile nature of plants subjects them to a constant exposure of biotic and abiotic stresses. Although plants do not have specialized immune cells, they can mount local and systemic immune responses, which require extensive crosstalk between plant defense and other physiological processes [1]. Induction of local defense responses involves recognition of microbe-associated molecular patterns (MAMPs) by membrane-associated receptors, leading to MAMP-triggered immunity (MTI), and recognition of pathogen-delivered effectors by cytosolic receptors, resulting in effector-triggered immunity (ETI) [2]. Salicylic acid (SA) that is produced during local infection events can lead to systemic acquired resistance (SAR). In Arabidopsis, SA signals through a key immune regulator, designated NPR1 (Non-expressor of PR genes), which is involved in regulating changes at the transcriptional level of as many as ˜10% of all genes [3]. Systemic acquired resistance is broad-spectrum and long lasting, compared to the signal-specific MAMP- and effector-triggered immunity responses [4].

SAR-associated transcriptional reprogramming re-directs cellular resources, normally dedicated to growth-related activities, towards de novo synthesis of anti-microbial proteins, such as the pathogenesis-related (PR) proteins. Before PR proteins can accumulate, endoplasmic reticulum (ER)-resident genes encoding the secretory pathway components are coordinately up-regulated to ensure efficient post-translational modification and secretion of the antimicrobial PR peptides [3, 5]. The enhancement of ER components is not restricted to SAR, however, as ER-resident genes have been shown to be involved in MTI. In studies directed to the biogenesis of EFR, a membrane-bound receptor for the MAMP signal elf18 (the N terminal 18 amino acids of the bacterial translation elongation factor Tu, EF-Tu), TBF1 was found to regulate glycosylation pathway genes, including calreticulin 3 (CRT3), and UDP-glucose:glycoprotein glycosyltransferase, STT3A, involved in the ER quality control mechanism (ERQC) required for EFR function [6, 7].

In earlier studies, we demonstrated that induction of both PR and ER-resident genes requires NPR1, a transcription cofactor. Upon induction by SA, NPR1 is translocated to the nucleus [8] inducing PR genes through its interaction with TGA transcription factors (TFs) at the promoters of PR genes [9, 10]. It is not known how NPR1 regulates the ER-resident genes. TGA TFs are not likely candidates, because expression of ER-resident genes is unaltered following induction in tga mutants [3]. Significant enrichment of a novel cis-element TL1 (translocon 1; GAAGAAGAA) in the promoter regions of these NPR1-dependent ER-resident genes suggests the involvement of an unknown TF [3]. Point mutations in the TL1 elements in the BiP2 (Lumenal Binding Protein 2) promoter abolished the inducibility of this gene upon SA treatment, supporting this hypothesis [3]. Identification of the TL1-binding TF is important to our understanding of the mechanism controlling the transition from growth- to defense-responses, as the secretory pathway is required for a wide variety of other cellular functions.

In this study, we report the identification of a heat shock factor-like protein (HSF4/HsfB1) that binds to the TL1 cis-element, which transcriptionally-regulates the expression of genes containing this motif in their promoter regions. We renamed it TL1-Binding Transcription Factor 1, TBF1, since mutants of this transcription factor have normal heat shock responses, but are compromised in the growth-to-defense transition upon challenge by pathogens. The translation of TBF1 is also tightly-regulated through two upstream open reading frames (uORFs) enriched in aromatic amino acids, which are precursors of a large array of plant secondary metabolites involved in defense. Taken together, these observations suggest that TBF1 plays a key role in the general control of events at the transcriptional level in plants.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to an isolated nucleic acid molecule comprising a regulatory region used to modulate the expression of one or more polypeptides in a cell, wherein said regulatory region comprises a promoter, functional in said cell, operably-linked to at least one upstream open reading frame (uORF) that encodes a polypeptide selected from the group consisting of: (a) (i) a polypeptide represented by uORF1 (SEQ ID NO: 102); (ii) a variant polypeptide thereof that contains one or more conservative substitutions in which one or more uORF1 functions are conserved; or (iii) a variant polypeptide thereof that contains one or more substitutions, fusions, or truncations in which one or more uORF1 functions are conserved; and (b) (i) a polypeptide represented by uORF2 (SEQ ID NO: 103); (ii) a variant polypeptide thereof that contains one or more conservative substitutions in which one or more uORF2 functions are conserved; or (iii) a variant polypeptide thereof that contains one or more substitutions, fusions, or truncations in which one or more uORF2 functions are conserved.

Separate aspects of the invention relate to a vector, cell, or a transgenic plant comprising a regulatory region used to modulate the expression of one or more polypeptides in a cell, wherein said regulatory region comprises a promoter, functional in said cell, operably-linked to at least one upstream open reading frame (uORF) that encodes a polypeptide selected from the group consisting of: (a) (i) a polypeptide represented by uORF1 (SEQ ID NO: 102); (ii) a variant polypeptide thereof that contains one or more conservative substitutions in which one or more uORF1 functions are conserved; or (iii) a variant polypeptide thereof that contains one or more substitutions, fusions, or truncations in which one or more uORF1 functions are conserved; and (b) (i) a polypeptide represented by uORF2 (SEQ ID NO: 103); (ii)a variant polypeptide thereof that contains one or more conservative substitutions in which one or more uORF2 functions are conserved; or (iii) a variant polypeptide thereof that contains one or more substitutions, fusions, or truncations in which one or more uORF2 functions are conserved.

Still another aspect of the invention relates to a method of using a regulatory region to modulate the expression of one or more polypeptides in a cell, wherein said regulatory region comprises a promoter, functional in said cell, operably-linked to one or more upstream ORFs and one or more downstream ORFs encoding said one or more polypeptides, comprising the steps of: (a) introducing one or more nucleic acids comprising said regulatory region into a cell; (b) expressing one or more upstream ORFs and one or more downstream ORFs encoding one or more polypeptides for a period sufficient to modulate the amount or level of activity of at least one of the one or more polypeptides within the cell or in the cell culture medium obtained from said cell. Another aspect relates to a method, further comprising the step (c) of purifying at least one of said polypeptides from the cell comprising said regulatory region or from the cell culture medium obtained from said cell.

A better understanding of the invention will be obtained from the following detailed descriptions and accompanying drawings, which set forth illustrative embodiments that are indicative of the various ways in which the principals of the invention may be employed.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

FIG. 1 sets forth an illustration showing that HSF4 is the TL1-Binding TF, TBF1.

(Panels 1A and 1B) TBF1 (HSF4) binding to the TL1 cis-elements in the BiP2 promoter was detected in Y1H. Strain 1 carries the WT BiP2 promoter upstream of both the HIS3 and LacZ reporters and Strain 2 contains a mutant BiP2 promoter in the TL1 elements upstream of LacZ [3]. Strain 1 and Strain 2 were both transformed with either pDEST-AD TBF1 encoding TBF1-AD, or the empty vector, pDEST-AD. Yeast growth assays for the HIS3 locus were performed on selective media (SD-His-Ura-Trp) supplemented with increasing concentrations of 3-AT, and photographed four days later (Panel 1A). β-galactosidase reporter activity was measured using ONPG as the substrate (Panel 1B). Error bars represent standard deviation from three different technical replications. The experiments were repeated three times with similar results.

(Panel 1C) Electrophoretic mobility shift assays were performed using plant extracts from wild-type (WT) and the tbf1 mutant, with (+) and without (−) 6 hr-treatment with 1 mM SA. 40,000 cpm of the radioactive probe containing the TL1 element was mixed with 10 μg of protein extract in the presence (+) or absence (−) of the unlabeled WT (cold) or the mutant TL1 (mTL1) oligo (5 pmol/μL). The autoradiograph was developed 24 hrs after electrophoresis. The arrow marks the TBF-TL1 complex. Asterisks indicate non-specific binding. The experiment was repeated three times with similar results.

(Panel 1D) TBF1-GFP binding to the TL1 elements in the BiP2 promoter was measured by ChIP analysis after treatment with H₂O or 1 mM SA. The PCR amplicons designated 1 to 6 (gray boxes in the upper panel) used in the ChIP analysis are shown, with TL1 elements highlighted in white. The arrow represents the translational start site of BiP2. After ChIP analysis using an antibody directed against GFP, the fold-enrichment for each amplicon was calculated from the real-time PCR results, which were normalized to input, and represented by the ratio between TBF1p:TBF1-GFP (in tbf1) and untransformed control plants (lower panel). Error bars represent the standard deviation from three different replicates. The experiment was repeated five times with similar results.

FIG. 2, related to FIGS. 1A and 1B, sets forth an illustration showing the yeast strains used in the Y1H assay.

(Panel 2A) BiP2 promoter containing multiple functional TL1 cis-elements (top) (SEQ ID NO: 95) and mutated TL1 (mTL1; bottom) (SEQ ID NO: 96) are shown.

(Panel 2B) Strain 1 contains the WT BiP2 promoter fragment upstream of the HIS3 and the LacZ reporters. Strain 2 contains the WT BiP2 promoter upstream of HIS3, but mTL1 upstream of LacZ.

FIG. 3, related to FIG. 1C, sets forth an illustration showing that TBF1 transcript levels in the tbf1 T-DNA insertion mutant.

(Panel 3A) Schematic representation of the T-DNA insertion site in the tbf1 mutant. The genomic organization of TBF1 encompassing exon 1, intron 1 and exon 2 is shown. The position and direction of the T-DNA insertion within TBF1 are indicated. Disruption of TBF1 leads to the loss-of-function mutant, designated as SALK_104713, also referred to as tbf1.

(Panel 3B) Expression of TBF1 in WT and the tbf1 mutant. Relative TBF1 transcript levels were determined by quantitative RT-PCR using cDNA generated from leaves of 3-week-old WT and tbf1 plants. The expression values were normalized using those of UBQ5 as internal standards. Error bars represent the standard deviation among three technical replications. The experiment was repeated three times with similar results.

FIG. 4, related to FIG. 1D, sets forth an illustration showing that TBF1p:TBF1-GFP can complement the tbf1 mutation.

Bacterial growth was quantified 3 days after infection with Psm ES4326 (OD_(600nm)=0.0001). Error bars represent the 95% confidence intervals determined from six replicates. The experiment was performed three times with similar results.

FIG. 5 sets forth an illustration showing that TBF1 Plays a Major Role in Transcriptional Reprogramming during MTI and SAR.

(Panels 5A and 5B) Relative transcript levels of secretory pathway genes were determined by qRT-PCR using cDNA generated from WT, tbf1 and npr1-1 plants treated with 1 mM SA. The expression levels of BiP2 and CRT3 carrying TL1 in their promoters (2A) and BiP3 and CRT1 without TL1 in their promoters (2B) were normalized to the transcript levels of the constitutively-expressed UBQ5. Error bars represent the standard deviation from nine technical replicates derived from three independent experiments.

(Panel 5C) The Venn diagram shows the numbers of TBF1-dependent SA down-regulated (SA down), SA up-regulated (SA up), elf18 up-regulated (elf18 up) and elf18 down-regulated (elf18 down) genes (p-value<0.05).

(Panels 5D and 5E) Heatmaps of TBF1-regulated genes in total numbers (top), degrees of TBF1 dependency (middle), and numbers of TL1 cis-elements in the gene promoters (bottom), in response to SA (2D) and elf18 (2E) treatments. Top-ranked functional groups were determined using DAVID Gene Ontology (GO) analysis for TBF1-dependent, SA-repressed or induced genes (2D), and elf18-repressed or induced genes (2E). The scale indicates the log-transformed p-values of down-(blue) and up-(yellow) regulated genes (top). Yellow lines indicate TBF1-dependency (middle), and yellow lines correspond to the numbers of TL1 cis-elements in the gene promoters (bottom).

FIG. 6, related to FIG. 5A, sets forth an illustration showing that SA-induced accumulation of ER chaperones BiP1/2 is affected in the tbf1 mutant.

Total protein extract was obtained from six leaves derived from three plants per genotype 6 hours after treatment with 1 mM SA. An accumulation of highly sequence-similar BiP1/2 proteins was detected on Western blots with an antibody directed against BiP (α-BiP). Ponceau S stain was used to verify equal loading amounts. The experiment was repeated three times with similar results.

FIG. 7, related to FIG. 5D, sets forth an illustration showing validation of the microarray data using qRT-PCR analysis of selected genes.

qRT-PCR analysis of selected TBF1-dependent SA-induced genes, identified in the microarray analysis. Leaves of 3-week-old Arabidopsis plants were sprayed with 1 mM SA or water (NT) and tissues collected 6 hrs later. The expression values were normalized using those of UBQ5 as the internal standards. The error bars represent the standard deviation among three technical replications. The experiment was repeated three times with similar results.

FIG. 8, related to FIG. 5D, sets forth an illustration showing validation of the microarray data using qRT-PCR analysis of selected genes.

qRT-PCR analysis of selected TBF1-dependent SA-repressed genes, identified in the microarray analysis. Leaves of 3-week-old Arabidopsis plants were sprayed with 1 mM SA or water (NT) and tissues collected 6 hrs later. The expression values were normalized using those of UBQ5 as the internal standards. Error bars represent the standard deviation among three technical replications. The experiment was repeated three times with similar results.

FIG. 9, related to FIG. 5E, sets forth an illustration showing validation of the microarray data using qRT-PCR analysis of selected genes.

qRT-PCR analysis of selected TBF1-dependent elf18-induced genes, identified in the microarray analysis. Leaves of 3-week-old Arabidopsis plants were infiltrated with 10 μM elf18 or water (NT) and tissues collected 2 hrs later. The expression values were normalized using those of UBQ5 as the internal standards. Error bars represent the standard deviation among three technical replications. The experiment was repeated three times with similar results.

FIG. 10, related to FIG. 5E, sets forth an illustration showing validation of the microarray data using qRT-PCR analysis of selected genes.

qRT-PCR analysis of selected TBF1-dependent elf18-repressed genes, identified in the microarray analysis. Leaves of 3-week-old Arabidopsis plants were infiltrated with 10 ρM elf18 or water (NT) and tissues collected 2 hrs later. The expression values were normalized using those of UBQ5 as the internal standards. Error bars represent the standard deviation among three technical replications. The experiment was repeated three times with similar results.

FIG. 11 sets forth an illustration showing that TBF1 is a Major Molecular Switch for the Growth-to-Defense Transition

(Panel 11A) Fresh weight of ten seedlings grown for 10 days on plates with MS growth media (Ctrl), MS supplemented with increasing concentrations of SA or 10 μM elf18. Error bars represent the standard deviation of three replicates. This experiment was repeated three times with similar results. Statistical analysis was performed using the Student's t-test, *, p-value<0.05, **, p-value<0.01, ***, p-value≤0.001.

(Panel 11B) Seedling recovery after a two-day treatment with the UPR inducer tunicamycin at 300 μg/L was measured 10 days later by counting the percentage of surviving seedlings (left), and by phenotype observations (right). Error bars represent standard deviation of three replicates. This experiment was repeated five times with similar results. Statistical analysis was performed using the Student's t-test, ***, p-value≤0.001.

(Panel 11C) Intracellular wash fluid (IWF) and total protein extracts from leaves of three-week-old WT, tbf1, tbf1 transformed with the WT TBF1 gene (TBF1 comp.), npr1-1, and bip2 dad2 were collected 24 hrs after 1 mM SA treatment and subsequently subjected to Western blotting using an antibody directed against PR1 (α-PR1). For loading controls, an antibody against tubulin (α-Tub) was used to probe the total protein blot.

(Panel 11D) Enhanced disease susceptibility was measured in 3-week-old WT, tbf1, TBF1 comp. and npr1-1 plants three days after infiltration with a bacterial suspension of Psm ES4326 (OD_(600nm)=0.0001). Error bars represent the 95% confidence intervals of twenty-four replicates derived from three independent experiments. This experiment was repeated at least five times with similar results. Statistical analysis was performed using the Bonferroni post-test, ***, p-value<0.0001.

(Panel 11E) SA-induced resistance was determined according to the schematic representation (upper panel) and the growth of Psm ES4326 was plotted as in (D) but with a higher initial inoculum (OD_(600nm)=0.001) (lower panel). Error bars represent 95% confidence intervals of twenty-four replicates derived from three independent experiments. Statistical analysis was performed using Bonferroni post-test, ***, p-value<0.0001.

(Panel 11F) elf18-induced resistance was measured according to the schematic representation (upper panel) and with the initial Psm ES4326 inoculum of OD_(600nm)=0.001 (lower panel). Error bars represent 95% confidence intervals of twenty-four replicates derived from three independent experiments. Statistical analysis was performed using Bonferroni post-test,***, p-value<0.0001.

FIG. 12, related to FIG. 11A, sets forth an illustration showing that the tbf1 mutant displays wild type levels of sensitivity to flg22.

Fresh weight of ten seedlings grown for 10 days on plates with regular MS growth media (−flg22) or MS supplemented with 10 μM flg22 (+flg22). Error bars represent standard deviation of three replicates. The experiment was repeated three times with similar results.

FIG. 13, related to FIG. 11, sets forth an illustration showing that the tbf1 mutant shows normal heat shock response.

Relative BiP2 transcript levels were determined by quantitative RT-PCR using cDNA generated from leaf tissue of room temperature (RT)-incubated and heat-shocked (at 37° C. for 2 hrs)) 3-week-old WT and tbf1 plants. The expression values were normalized using those of UBQ5 as the internal standards. Error bars represent the standard deviation among three technical replications. Experiment was repeated three times with similar results.

FIG. 14, related to FIG. 11C, sets forth an illustration showing that PR1 transcript levels are not altered in the tbf1 mutant.

Relative PR1 transcript levels were determined by quantitative RT-PCR using cDNA generated from leaf tissue of 3-week-old WT, tbf1 and npr1-1 plants. Samples were harvested at 0 and 16 hrs after 1 mM salicylic acid (SA) application. The expression values were normalized using those of UBQ5 as the internal standards. Error bars represent the standard deviation among three technical replications. The experiment was repeated three times with similar results.

FIG. 15, related to FIG. 11F, sets forth an illustration showing that the tbf1 mutant has normal flg22-induced resistance to Psm ES4326.

(Panel 15A) Leaves were first injected with H₂O or 10 μM flg22 4 hrs prior to bacterial infection. Disease symptoms upon infection with Psm ES4326 (OD_(600nm)=0.001) were observed at 3.5 days post inoculation.

(Panel 15B) Leaves were first treated with H₂O or 10 μM flg22 for 4 hrs followed by infection with Psm ES4326 (OD_(600nm)=0.001). Bacterial growth was quantified at 3.5 days post inoculation. Error bars represent the 95% confidence interval of eight replicates. The experiment was performed three times with similar results.

FIG. 16 sets forth an illustration showing that TBF1 Expression is Regulated at both the Transcriptional and Translational Levels.

(Panels 16A and 16B) Relative transcript levels of TBF1 (16A) and NPR1 (16B) genes in response to 1 mM SA treatment were determined by qRT-PCR using cDNA generated from WT, tbf1 and npr1-1 plants. The expression values were normalized using the transcript levels of UBQ5. Error bars represent standard deviation from nine technical replicates derived from three independent experiments.

(Panel 16C) Schematic representation of uORF1 and uORF2 and exon I of TBF1. The phenylalanines (F) in uORF1 and uORF2 are highlighted in red and the stop codons are shown as asterisks. “+1” represents the translational start of TBF1 and −451, −266, and −217 represent the upstream positions of the 5′ end of the transcript, the start codon for uORF1 and the start codon for uORF2, respectively.

(Panel 16D) The effects of uORFs on TBF1 translation were determined by transiently expressing uORF1-uORF2-GUS (WT), uorf1-uORF2-GUS, uORF1-uorf2-GUS and uorf1-uorf2-GUS constructs under the control of the 35S promoter in Nicotiana benthamiana leaves, followed by GUS activity quantification 3 days later. GUS activities from mutant constructs were normalized to that of the WT construct. This experiment was repeated three times with similar results.

(Panel 16E) Quantification of translational inhibitory effect exerted by uORFs in transgenic T₃ plants expressing uORF1-uORF2-GUS (two independent transformants 6-1 and 9-4) or uorf1-uorf2 GUS (two independent transformants 7-3 and 8-3) at various time points after inoculation with Psm ES4326/avrRpt2 (OD_(600nm)=0.02). Error bars represent the standard deviation from three different replicates. The experiment was repeated at least three times with similar results.

(Panel 16F) Polysome profiles (upper panel) and TBF1 expression (lower panel) in samples obtained from WT plants at 0, 0.5 and 1 hr after inoculation with Psm ES4326/avrRpt2 (OD_(600nm)=0.02). The fractions containing monosome and polysome were identified based on the absorbance at 254 nm (A_(254nm)). The TBF1 transcript abundance normalized against Alien Alert® control transcript is expressed in arbitrary units (AU). Error bars represent standard error. This experiment was repeated using two biological replicates (each with three technical replicates) with similar results.

FIG. 17 sets forth an illustration showing that TBF1 Translation is Regulated in Response to Pathogen-Induced Changes in Phenylalanine Metabolism.

(Panel 17A) The effects of phenylalanine and aspartate starvation on the translational inhibitory function of uORFs were measured by growth of the yeast strain aro7 (phe-, tyr-) transformed with the uORF1-uORF2-DHFR or DHFR reporter. 80 μM methotrexate was added to the media so that yeast growth became dependent on the DHFR reporter expression. Optical densities for cultures containing two different concentrations of phenylalanine (Phe; 15 and 75 mg/L) and for cultures lacking Asp, but supplemented with 15 mM tobramycin (TOB), an inhibitor of yeast tRNA^(Asp) aspartylation, were recorded over the course of 32 hrs. Error bars represent the standard deviation from nine technical replicates derived from three independent experiments.

(Panel 17B) tRNA analysis of wild type plants 0, 0.5, 1, 2, 3, 4, and 8 hrs after inoculation with Psm ES4326/avrRpt2 (OD_(600nm)=0.02). tRNA was extracted from leaf samples, and a Northern blot experiment using DIG-labeled probes (Roche Applied Science) against tRNA^(Phe) or tRNA^(Asp) was performed to detect charged and uncharged tRNA^(Phe) or tRNA^(Asp). This experiment was repeated using three biological replicates with similar results.

(Panel 17C) Total protein extracts from leaves of three-week-old WT plants were collected at various time points after inoculation with Psm ES4326/avrRpt2 (OD_(600nm)=0.02) and subsequently subjected to Western blotting analysis using an antibody directed against a phosphorylated form of eIF2α (peIF2α, Epitomics). Ponceau S stain was used to determine the sample amounts needed for equal loading.

(Panel 17D) A model illustrating the molecular mechanism by which the translation initiation of TBF1 is regulated through rapid increases in uncharged and charged tRNA^(Phe), phosphorylation of eIF2α, and ribosomal read-through of uORFs.

FIG. 18, related to FIG. 17A, sets forth an illustration showing that the uORF1-uORF2-DHFR and DHFR recombinant proteins do not affect yeast growth in the absence of methotrexate.

The growth of yeast aro7 strains (Phe, Tyr) carrying uORF1-uORF2-TBF1_(1st exon)-DHFR (uORF1-uORF2-DHFR) or DHFR in the absence of methotrexate was measured over the course of ˜47 hrs by optical density (OD_(600nm)). The selective media (SD-Leu-Phe) was supplemented with 15 mg/L (Phe 15) and 75 mg/L (Phe 75) of phenylalanine, respectively. Error bars represent the standard deviation from three technical replicates. This experiment was repeated three times with similar results.

FIGS. 19 and 20 display the sequence and genetic elements of the TBF1 region in compact form

FIG. 19, continued as FIG. 20, displaying the sequence and genetic elements of the TBF1 region set forth in SEQ ID NO: 101 in compact form.

FIG. 21 presents quantification of translational inhibitory effect exerted by uORFs in transgenic T₃ plants

FIG. 21 presents quantification of translational inhibitory effect exerted by uORFs in transgenic T₃ plants expressing uORF1-uORF2-GUS or uorf1-uorf2-GUS in Ler or gcn2 backgrounds (two independent lines per construct per genotype) at indicated time points after inoculation with Psm ES4326/avrRpt2. Error bars represent the standard deviation from three technical replicates.

FIG. 22 sets forth a schematic diagram of TBF1 mRNA encoding uORF1 and uORF1.

The potential peptides encoded by uORF1 (SEQ ID NO: 102) and uORF2 (SEQ ID NO: 103) are shown at the bottom of the illustration.

FIG. 23 sets forth a schematic diagram of the expression cassettes.

Target genes can be inserted to replace the Gateway cassette using the adapters LIC1 (SEQ ID NO: 130) and LIC2 (SEQ ID NO: 131). The 5′UTR of TBF1 with native uORFs (starting with an ATG codon, pGX1 (SEQ ID NO: 132)/pGX180 (SEQ ID NO: 135)) or mutant uorfs (starting with a CTG codon, pGX181 (SEQ ID NO: 133)/pGX179 SEQ ID NO: (134)) are placed upstream of the Gateway cassette. The 35S promoter with duplicated enhancers (pGX179 (SEQ ID NO: 134)/pGX180 (SEQ ID NO: 135)) or the TBF1 promoter (pGX1 (SEQ ID NO: (132)/pGX181 (SEQ ID NO: 133)) is used to drive expression of downstream sequences. The genetic elements are as follows: TBF1 pro: TBF1 promoter; 35S Pro: 35S promoter with duplicated enhancers; uORF1/2: upstream open reading frame; uorf1/2: mutant form of uORF1/2 respectively; LIC1/2: ligation-independent cloning sequences; NOS; NOS terminator.

FIG. 24 sets forth an illustration demonstrating that TBF1 uORF suppresses both cytosol-synthesized and ER (endoplasmic reticulum)-synthesized proteins.

Genes encoding luciferase (synthesized in the cytosol) and mGFP5 (synthesized in the ER) are cloned into pGX179 (SEQ ID NO: 134) and pGX180 (SEQ ID NO: 135) as 35S::uORF-Luciferase/35S::uorf-Luciferase and 35S::uORF-mGFP5/35S::uorf-mGFP5, respectively. Luciferase activity of transgenic Arabidopsis seedlings harboring the 35S::uORF-Luciferase cassette (left) or the 35S::uorf-Luciferase cassette (right) are detected by CCD camera after the application of luciferin substrate. Agrobacteria containing the 35S::uORF-mGFP5 cassette (left) or the 35S::uorf-mGFP5 cassette (right) were injected into N. benthamiana. N. benthamiana leaves were observed under UV at two days post-injection. Red fluorescence is observed in chloroplasts. These results demonstrate that the uORF region can suppress both the activity of luciferase and the level of mGFP in the transformed plant cells.

TERMS AND ABBREVIATIONS

The following is a list of terms and their definitions used throughout the specification and the claims:

The terms “cell” and “cells”, which are meant to be inclusive, refer to one or more cells which can be in an isolated or cultured state, as in a cell line comprising a homogeneous or heterogeneous population of cells, or in a tissue sample, or as part of an organism, such as an unmodified or a transgenic plant or animal.

General abbreviations and their corresponding meanings include: aa or AA=amino acid; mg=milligram(s); ml or mL=milliliter(s); mm=millimeter(s); mM=millimolar; nmol=nanomole(s); pmol=picomole(s); ppm=parts per million; RT=room temperature; U=units; ug, μg=micro gram(s); ul, μl=micro liter(s); uM, μM=micromolar; HPLC, high-performance liquid chromatography; ORF=open reading frame; PCR=polymerase chain reaction; SDS-PAGE=sodium dodecyl sulfate-polyacrylamide gel electrophoresis; RT=reverse transcriptase.

DETAILED DESCRIPTION OF THE INVENTION

Induction of plant immune responses involves significant reprogramming of transcription that prioritizes defense-over growth-related cellular functions. Despite intensive efforts involving forward genetic screens and genome-wide expression-profiling experiments, only a limited number of transcription factors have been found that are involved in regulating the growth-to-defense transition. Using endoplasmic reticulum (ER)-resident genes required for antimicrobial protein secretion as markers, we identified a heat shock factor-like transcription factor that specifically binds to the TL1 (GAAGAAGAA) cis-element required for the induction of these genes. Plants lacking this TL1-binding factor (TBF1) respond normally to heat stress, but were shown to be compromised in their immune responses induced by salicylic acid (SA), and by microbe-associated molecular pattern (MAMP), elf18. Genome-wide expression profiling indicated that TBF1 plays a key role in the growth-to-defense transition. The expression of TBF1 itself was shown to be tightly regulated at both the transcriptional and translational levels. Two small upstream open reading frames (uORFs) encoding multiple aromatic amino acids were found 5′ to the translation initiation codon of TBF1 and shown to affect its translation. Through this unique regulatory mechanism, TBF1 can sense metabolic changes upon invasion by pathogens, triggering specific transcriptional reprogramming by modifying the expression of its target genes. Key aspects of this study can be summarized as follows: (1) the plant transcription factor, TBF1, binds to the TL1 element in vitro and in vivo; (2) TBF1 controls the expression of nearly 3,000 genes involved in development and immunity; (3) TBF1 is required for effective SA- and MAMP-induced defense responses; and (4) translation of TBF1 is regulated by uORFs in 5′ UTR and is sensitive to metabolic changes.

The present invention relates to an isolated nucleic acid molecule comprising a regulatory region used to modulate the expression of one or more polypeptides in a cell, wherein said regulatory region comprises a promoter, functional in said cell, operably-linked to at least one upstream open reading frame (uORF) that encodes a polypeptide selected from the group consisting of: (a) (i) a polypeptide represented by uORF1 (SEQ ID NO: 102); (ii) a variant polypeptide thereof that contains one or more conservative substitutions in which one or more uORF1 functions are conserved; or (iii) a variant polypeptide thereof that contains one or more substitutions, fusions, or truncations in which one or more uORF1 functions are conserved; and (b) (i) a polypeptide represented by uORF2 (SEQ ID NO: 103); (ii) a variant polypeptide thereof that contains one or more conservative substitutions in which one or more uORF2 functions are conserved; or (iii) a variant polypeptide thereof that contains one or more substitutions, fusions, or truncations in which one or more uORF2 functions are conserved.

An aspect of the invention relates to an isolated nucleic acid, as described above, wherein said molecule comprises uORF1, or a functional variant thereof, and uORF2, or a functional variant thereof. Another aspect, relates to an isolated nucleic acid, further comprising one or more downstream ORFs (dORFs) encoding one or more polypeptides. In another aspect, at least one dORF encodes a polypeptide selected from the group consisting of: (i) a polypeptide that is functionally-active as a transcription factor; (ii) a reporter polypeptide; (iii) a polypeptide that confers resistance to drugs or agrichemicals; (iv) a polypeptide involved in in resistance of plants to viral, bacterial, fungal pathogens, oomycete pathogens, phytoplasmas, and nematodes; and (v) a polypeptide involved in the growth or development of plants.

A variety of polypeptides encoded by a downstream ORF are contemplated by the invention. In one aspect, the polypeptide is a transcription factor selected from the group consisting of: (i) a polypeptide represented by TBF1 (SEQ ID NO: 106); (ii) a variant polypeptide thereof, that contains one or more conservative substitutions in which one or more TBF1 functions are conserved; or (iii) a variant polypeptide thereof, that contains one or more substitutions, fusions, or truncations in which one or more TBF1 functions are conserved. In another aspect, the polypeptide is a reporter polypeptide selected from the group consisting of: (i) β-galactosidase (β-gal), β-glucuronidase (β-gluc), chloramphenicol acetyltransferase (CAT), Renilla-luciferase (ruc), Photinus luciferase (luc), secreted alkaline phosphatase (SAP), and green fluorescent protein (GFP); (ii) a variant of the reporter polypeptide specified in (i) that contains one or more conservative substitutions in which one or more reporter functions are conserved; or (iii) a variant of the reporter polypeptide specified in (i) that contains one or more substitutions, fusions, or truncations in which one or more reporter functions are conserved.

The invention is not limited by the specific nature of the polypeptide encoded by the downstream ORF, provided it is functional in the cellular or organismal environment being evaluated. In some cases, it may be desirable to express a partially-functional or non-functional polypeptide, compared to a fully-functional polypeptide to study its properties with respect to its biological activity, including its binding affinity to, or influence on the properties of, other cellular molecules. In this respect, polypeptides being studied, including those encoded by upstream or downstream ORFS (uORFs and dORFs), may contain a variety of alterations, such as conservative substitutions, in which amino acids having similar structural or chemical properties (e.g., size, charge, or polarity) are substituted for amino acids in the unmodified polypeptide. A variety of polypeptides can tolerate insertions of other polypeptide segments, at the amino terminus, carboxy terminus, or at internal positions, permitting the evaluation of protein fusions, which may retain or interfere with the activity of the unmodified polypeptide. Many polypeptides can also tolerate internal deletions, or truncations of amino acids at the amino terminus or carboxy terminus, which may retain or interfere with the activity of the unmodified polypeptide. Polypeptides may also contain one or more alterations, such as substitutions, insertions/fusions, deletions/truncations, in a variety of combinations, which alter the structure, and in some cases function, of the polypeptide being studied.

The types of alterations that are tolerated depend on the nature of the polypeptide being studied. For example, for polypeptides having more than one function, alterations may be tolerated in specific structural domains, if the system being evaluated is not sensitive to the function carried out by polypeptide domain. Reporter polypeptides, for example, may more easily tolerate alterations at either end of the polypeptide, permitting the construction of fused or truncated polypeptides, that retain the catalytic activity responsible for the reporter function (e.g., enzymatic activity, or fluorescence), than alterations located in the middle of the molecule. Transcriptional factors, like TBF1, may tolerate alterations in regions that are not involved in the binding of the polypeptide to nucleic acids, other polypeptides, or other types of regulatory co-factors.

The promoters used with the invention may comprise a variety of genetic elements that regulate their properties, including level of transcription at different times, generally in response to different concentrations of general or specific transcriptional components, including regulatory molecules, polymerase complexes, typically be small molecules, nucleic acids, peptides, or polypeptides, or conjugates between these and other cellular molecules or macromolecules. In one aspect of the invention, the promoter is constitutive, and in another aspect, the promoter is inducible.

In one aspect, the promoter is active in plant cells. In one aspect, the promoter is selected from the group consisting of: (a) a plant promoter; (b) a plant virus promoter; (c) a promoter from a non-viral plant pathogen; (d) a mammalian cell promoter; and (e) a mammalian virus promoter. In one aspect, the promoter is a plant promoter. In another aspect, the plant promoter is selected from the group consisting of: (a) the TBF1 promoter as set forth in SEQ ID NO: 113; (b) a variant sequence thereof, that contains one or more substitutions, insertions, or deletions, in which one or more TBF1 promoter functions are preserved; or (c) a nucleotide sequence which is 50% or more identical to the TBF1 promoter set forth in (a) in which one or more promoter functions are preserved. In another aspect, the plant promoter is selected from the group consisting of: (a) the BiP2 promoter as set forth in SEQ ID NO: 109; (b) a variant sequence thereof, that contains one or more substitutions, insertions, or deletions, in which one or more TBF1 promoter functions are preserved. In another aspect, the plant promoter is a nucleotide sequence comprising a binding site for the TBF1 polypeptide in which one or more promoter functions are preserved. In a specific aspect, the plant promoter is a nucleotide sequence comprising a functionally-active pathogen-inducible or constitutive promoter. In more specific aspect, the promoter is derived from an Arabidopsis locus selected from the group consisting of AT1G48850, AT1G62300, AT4G34230, AT4G34180, AT4G35110, AT2G30490, AT5G38900, AT5G24430, AT1G63720, AT4G39270.

In one aspect, the promoter is a plant promoter which is inducible. In another aspect, the plant promoter is inducible upon challenge by a plant pathogen or a chemical inducer. In another aspect, the inducer is selected from the group consisting of salicylic acid, jasmonic acid, methyl ester of jasmonic acid, abscisic acid, ethylene, AgNO₃, cycloheximide, mannitol, NaCl, flg22, elf18 and LPS. This non-limiting list of inducers have all been tested and shown to induce the TBF1 promoter. Other stimuli, which trigger a similar induction response in TBF1-like genes could be used to test their ability to modulate expression mediated the regulatory region described above.

Other aspects of the invention include cells and vectors comprising nucleic acids comprising the regulatory regions described above, and organisms, particularly plant propagation material, plants, and seeds derived from plants comprising said cells or vectors. One aspect, for example, is a cell comprising a nucleic acid with a regulatory region comprising a promoter operable in said cell, and one or more upstream ORFs, optionally linked to one or more downstream ORFS, as described above. In another aspect, the cell is a plant cell and said promoter is active in plant cells. Another aspect is plant propagation material comprising said cell. Other aspects include a transgenic plant comprising said cell, and a seed derived from said transgenic plant.

Related aspects include a vector comprising a nucleic acid with a regulatory region comprising a promoter operable in a cell, and one or more upstream ORFs, optionally linked to one or more downstream ORFS, as described above. Another aspect is a cell comprising a vector comprising the regulatory region as noted above, and a plant cell comprising the vector, wherein the promoter is active in plant cells. Other aspects include a transgenic plant comprising the vector, and the seed of a transgenic plant comprising the vector described above.

It should be noted that vectors may carry genetic elements, such as those that confer resistance to drugs, that are not essential to the function of the nucleic acids of the invention that comprise the regulatory region (e.g., promoter, one or more uORFs, optionally one or more dORFs) described above. The vectors may be plasmids, propagated in bacteria or plants, or viruses. Plasmids are typically propagated as double-stranded DNA circles, while viruses may carry genetic information as single- or double-stranded RNA or DNA molecules. The nucleic acids that comprise the regulatory region noted above may be introduced into cells as part of a larger molecule, such as a vector, or introduced directly into a cell not covalently linked to other nucleic acids, although other nucleic acids or vectors may be used to facilitate the introduction of genetic material, such as selectable or screenable genetic markers, into the cell. The nucleic acids of the invention, therefore, may not be stably-propagated, after introduction into a cell, or may be stably-propagated, either by replication of a vector comprising the regulatory region noted above, or by stable integration of the nucleic acid at one or more regions within the genome of the cell.

One aspect of the invention relates to a transgenic plant comprising a regulatory region used to modulate the expression of one or more polypeptides in a cell, wherein said regulatory region comprises a promoter, functional in said cell, operably-linked to at least one upstream open reading frame (uORF) that encodes a polypeptide selected from the group consisting of: (a) (i) a polypeptide represented by uORF1 (SEQ ID NO: 102); (ii) a variant polypeptide thereof that contains one or more conservative substitutions in which one or more uORF1 functions are conserved; or (iii) a variant polypeptide thereof that contains one or more substitutions, fusions, or truncations in which one or more uORF1 functions are conserved; and (b) (i) a polypeptide represented by uORF2 (SEQ ID NO: 103); (ii)a variant polypeptide thereof that contains one or more conservative substitutions in which one or more uORF2 functions are conserved; or (iii) a variant polypeptide thereof that contains one or more substitutions, fusions, or truncations in which one or more uORF2 functions are conserved. A related aspect includes a transgenic plant, wherein said molecule comprises uORF1, or a functional variant thereof, and uORF2, or a functional variant thereof.

Another aspect includes a transgenic plant further comprising one or more downstream ORFs (dORFs) encoding one or more polypeptides. The invention also includes a transgenic plant, wherein at least one dORF encodes a polypeptide selected from the group consisting of: (i) a polypeptide that is functionally-active as a transcription factor; (ii) a reporter polypeptide; (iii) a polypeptide that confers resistance to drugs or agrichemicals; (iv) a polypeptide involved in in resistance of plants to viral, bacterial, fungal pathogens, oomycete pathogens, phytoplasmas, and nematodes; and (v) a polypeptide involved in the growth or development of plants.

Related aspects of the invention also include transgenic plants wherein downstream ORFs encode specific polypeptides, such as TBF1, and natural or synthetic variants, homologues, and orthologs, or reporter polypeptides, such as β-glucuronidase, β-galactosidase, luciferase, and fluorescent proteins, as noted above.

The invention also relates to a variety of methods of using the regulatory region described above to facilitate the expression (e.g., transcription of mRNA, and translation of the mRNA comprising one or more ORFs) of one or more peptides or polypeptides in a cell. A polypeptide may be also released from the cell into the extracellular environment, such as cell culture medium, after being processed for secretion, or by degradation of the cell membrane or cell wall, where it may be recovered and purified. It is not necessary for a polypeptide to be expressed at high levels to have an effect on other cellular functions. A transcriptional factor, for example, may have pleiotropic effects by modulating its expression only slightly, compared to the amount or level of activity in a parent cell that does not contain a regulatory region described above.

One aspect of the invention relates to a method of using a regulatory region to modulate the expression of one or more polypeptides in a cell, wherein said regulatory region comprises a promoter, functional in said cell, operably-linked to one or more upstream ORFs and one or more downstream ORFs encoding said one or more polypeptides, comprising the steps of: (a) introducing one or more nucleic acids comprising said regulatory region into a cell; (b) expressing one or more upstream ORFs and one or more downstream ORFs encoding one or more polypeptides for a period sufficient to modulate the amount or level of activity of at least one of the one or more polypeptides within the cell or in the cell culture medium obtained from said cell. Another aspect relates to a method, further comprising the step (c) of purifying at least one of said polypeptides from the cell comprising said regulatory region or from the cell culture medium obtained from said cell.

In any of these methods, the amount or level of activity at least one of said polypeptides may be enhanced above, or reduced below, the endogenous amount or level of activity in a parent cell lacking an introduced nucleic acid comprising said regulatory region.

Another aspect relates to a method wherein said regulatory region contains a nucleic acid comprising at least one upstream open reading frame (uORF) that encodes a polypeptide selected from the group consisting of: (a) (i) a polypeptide represented by uORF1 (SEQ ID NO: 102); (ii) a variant polypeptide thereof that contains one or more conservative substitutions in which one or more uORF1 functions are conserved; or (iii) a variant polypeptide thereof that contains one or more substitutions, fusions, or truncations in which one or more uORF1 functions are conserved; (b) (i) a polypeptide represented by uORF2 (SEQ ID NO: 103); (ii) a variant polypeptide thereof that contains one or more conservative substitutions in which one or more uORF2 functions are conserved; or (iii) a variant polypeptide thereof that contains one or more substitutions, fusions, or truncations in which one or more uORF2 functions are conserved.

Related aspects include methods wherein said nucleic acid molecule comprises uORF1, or a functional variant thereof, and uORF2, or a functional variant thereof, and also methods wherein said nucleic acid molecule further comprises one or more downstream ORFs (dORFs) encoding one or more polypeptides.

Another aspect includes a method wherein the regulatory region further comprises one or more downstream ORFs (dORFs) encoding one or more polypeptides. The invention also includes a method wherein at least one dORF encodes a polypeptide selected from the group consisting of: (i) a polypeptide that is functionally-active as a transcription factor; (ii) a reporter polypeptide; (iii) a polypeptide that confers resistance to drugs or agrichemicals; (iv) a polypeptide involved in in resistance of plants to viral, bacterial, fungal pathogens, oomycete pathogens, phytoplasmas, and nematodes; and (v) a polypeptide involved in the growth or development of plants. Related aspects include methods where the downstream ORFs encode specific polypeptides, such as TBF1, and natural or synthetic variants, homologues, and orthologs, or reporter polypeptides, such as β-glucuronidase, β-galactosidase, luciferase, and fluorescent proteins, as noted above.

Other aspects also relate to methods wherein the regulatory region comprises a specific promoter, such as those described above, which may be constitutive or inducible, or derived from different sources, provided they are functionally active in the cell or organism being evaluated.

The invention is also directed to any of the methods described above that include introducing the nucleic acid comprising the regulatory region comprising a promoter, one or more uORFs, and one or more dORFs, into a cell, expressing a polypeptide under the control of the regulatory region, and purifying the polypeptide from a cell, tissue, or plants, or its extracellular environment.

While specific aspects of the invention have been described in detail, it will be appreciated by those skilled in the art that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure. Accordingly, the particular arrangements disclosed are meant to be illustrative only, and not limiting as to the scope of the invention, which is to be given the full breadth of the appended claims, and any equivalent, thereof.

EXAMPLES

The foregoing discussion may be better understood in connection with the following representative examples which are presented for purposes of illustrating the principle methods and compositions of the invention, and not by way of limitation. Various other examples will be apparent to the person skilled in the art after reading the present disclosure without departing from the spirit and scope of the invention. It is intended that all such other examples be included within the scope of the appended claims.

General Materials and Methods and Sources of Materials

All parts are by weight (e.g., % w/w), and temperatures are in degrees centigrade (° C.), unless otherwise indicated. Table 1 presents a summary of the PCR primers and nucleotide and amino acid sequences described in this application.

TABLE 1 PCR Primers, Nucleotide and Amino Acid Sequences and SEQ ID NOS Used In This Study SEQ ID Name Description Length Type NO: A. primers used in the ChIP experiment BiP2-ChIP-F1 ATGGCTCGGCTCGCT 15 ssDNA 1 BiP2-ChIP-R1 GAGATCAAGCAACAATGCAGA 21 ssDNA 2 BiP2-ChIP-F2 TCGGGCACTGGACCTATTTA 20 ssDNA 3 BiP2-ChIP-R2 CGGAAACTTTTGCGTACGAT 20 ssDNA 4 BiP2-ChIP-F3 GGCCACGATTACTCCAACAC 20 ssDNA 5 BiP2-ChIP-R3 TCGCTTTTTATGGAAGACGAA 21 ssDNA 6 BiP2-ChIP-F4 GGTTCCGGTTCTTTTCCACT 20 ssDNA 7 BiP2-ChIP-R4 TGTGTTGGAGTAATCGTGGC 20 ssDNA 8 BiP2-ChIP-F5 GGTACGCAGATCGGATTCGAGTAAAAC 27 ssDNA 9 BiP2-ChIP-R5 TTATAGCCAATTGATCCGAACCAAAACCG 29 ssDNA 10 BiP2-ChIP-F6 CATCCAAAAATATATTAGTACGAGCC 26 ssDNA 11 BiP2-ChIP-R6 CCATCACCGTTAACAAAGAAA 21 ssDNA 12 B. Primers used for real-time PCR UBQ5-qPCR-F GACGCTTCATCTCGTCC 17 ssDNA 13 UBQ5-qPCR-R GTAAACGTAGGTGAGTCCA 19 ssDNA 14 TBF1-qPCR-F GTTGGTTCGCCTTCTG 16 ssDNA 15 TBF1-qPCR-R CCACACCCCAAACAAT 16 ssDNA 16 BiP2-qPCR-F GACGCCAACGGTATTC 16 ssDNA 17 BiP2-qPCR-R TGTCTCCAGGGCATTC 16 ssDNA 18 CRT3-qPCR-F ATGACCCCAACGATGT 16 ssDNA 19 CRT3-qPCR-R CCTTGTAGTTCGGGTTCT 18 ssDNA 20 PR1-qPCR-F CTCATACACTCTGGTGGG 18 ssDNA 21 PR1-qPCR-R TTGGCACATCCGAGTC 16 ssDNA 22 BiP3-qPCR-F AGCACTCGAATCCCAA 16 ssDNA 23 BiP3-qPCR-R GCCTCCGACAGTTTCA 16 ssDNA 24 CRT1-qPCR-F CTGTGGTGGTGGCTAC 16 ssDNA 25 CRT1-qPCR-R GTCTCACATGGGACCT 16 ssDNA 26 NPR1-qPCR-F AAACATGTCTCGAATGT 17 ssDNA 27 NPR1-qPCR-R GATTCCTATGGTTGACA 17 ssDNA 28 CNX1-qPCR-F CCCATGTCTACACCGC 16 ssDNA 29 CNX1-qPCR-R CACGGCATTTGGATCAG 17 ssDNA 30 MPK11-qPCR-F ACCCAAACAGACGCATTACAG 21 ssDNA 31 MPK11-qPCR-R CTCCTTGATGTTCTCTTCCGTC 22 ssDNA 32 GWD-qPCR-F ATCGCAGATTTGGAGAGTGAG 21 ssDNA 33 GWD-qPCR-R TGTAGCCATAAACCTCATCCAG 22 ssDNA 34 TGA3-qPCR-F TCTTGATGTCGGGAATGTGG 20 ssDNA 35 TGA3-qPCR-R AGTTGCTGATCGGTTAAGGG 20 ssDNA 36 SecY-qPCR-F TTCTACACCTCCAACATGCC 20 ssDNA 37 SecY-qPCR-R CTCTGATTCTTTCCACTGTCCC 22 ssDNA 38 PR-13-qPCR-F TGTGTATCCTCTGTTTGCGG 20 ssDNA 39 PR-13-qPCR-R TGCATTCATAGAGCCCTTGG 20 ssDNA 40 CRR23-qPCR-F ACATCTCACACCAAACCCAAC 21 ssDNA 41 CRR23-qPCR-R TAAGGCTGGATGGTCAATCG 20 ssDNA 42 OB-fold-like-qPCR-F AACCTACCACGAACACCATC 20 ssDNA 43 OB-fold-like-qPCR-R ACTACATAAGCGGCCATCAG 20 ssDNA 44 ANNAT4-qPCR-F CAAACCAAGAGCCGGAAATC 20 ssDNA 45 ANNAT4-qPCR-R TCCCCAGTGTGCTTATCAATG 21 ssDNA 46 FLA8-qPCR-F TGCTCCACACTGACACTTG 19 ssDNA 47 FLA8-qPCR-R GCGAGGATTTGAGTGATGTTG 21 ssDNA 48 HSP70-qPCR-F AATGGCTGGTAAAGGAGAAGG 21 ssDNA 49 HSP70-qPCR-R CTATCAGTGAAGGCGACGTAAG 22 ssDNA 50 ATERF-7-qPCR-F AAATCTCGTGTCTGGCTCG 19 ssDNA 51 ATERF-7-qPCR-R AGGTGAGAGGTTGGAGAGG 19 ssDNA 52 DegP-qPCR-F AAGAGAACACTCCTTCCGTTG 21 ssDNA 53 DegP-qPCR-R TGACCTTGCTTATCCCACAC 20 ssDNA 54 Thylakoid P17-qPCR-F GAGATCCAGTTCCTTGTGAGAG 22 ssDNA 55 Thylakoid P17-qPCR-R ATTCCACCTTCATCTTCCCTTC 22 ssDNA 56 MPK3-qPCR-F CGAAAAGATACATCCGGCAAC 21 ssDNA 57 MPK3-qPCR-R GATTCAGAGCTTGTTCAACAGTG 23 ssDNA 58 Clathrin-qPCR-F CCTCGTGAAGTGCCAGTTATAG 22 ssDNA 59 Clathrin-qPCR-R GGATTGTGCTTGAGTTTCGTG 21 ssDNA 60 PAD4-qPCR-F AAGATCCATGACATCGCCG 19 ssDNA 61 PAD4-qPCR-R AGGTAGAGGTTCATCGGAGG 20 ssDNA 62 PDIL-qPCR-F GTGGATGTTGACCGTACAGTAG 22 ssDNA 63 PDIL-qPCR-R CTTGGAACTATCACCCTCGATC 22 ssDNA 64 PGL34-qPCR-F GCTTCTCATCCTCTGTATCACC 22 ssDNA 65 PGL34-qPCR-R AACCGAGTCTTGAACCATAGC 21 ssDNA 66 SAUR-like-qPCR-F TGGATTCGAGCAGAAAGGTAC 21 ssDNA 67 SAUR-like-qPCR-R TGGGTTAGGCCGTGTTTG 18 ssDNA 68 GA2ox-qPCR-F GAACCGATATCCACCTTGTCC 21 ssDNA 69 GA2ox-qPCR-R TGAGGAAATCACTGTCCGTG 20 ssDNA 70 EXLA2-qPCR-F TGACAAAGTACCCAACGGAG 20 ssDNA 71 EXLA2-qPCR-R ATGTCTGTGATCTGAACGCC 20 ssDNA 72 CESA6-qPCR-F TCCTTCTCGCCTCTATCCTTAC 22 ssDNA 73 CESA6-qPCR-R CAGTCCAAGCCACATATCTCG 21 ssDNA 74 WRKY75-qPCR-F GGAGGGATATGATAATGGGTCG 22 ssDNA 75 WRKY75-qPCR-R ACCTTCTGATCTAACCTTTGAGC 23 ssDNA 76 Ribosomal Protein-qPCR-F TTGCCTCTGAAATGAGTCCG 20 ssDNA 77 Ribosomal Protein-qPCR-R TGCTCTTCCCCTTTGTTCTC 20 ssDNA 78 MDH-qPCR-F TTGCCTCTGAAATGAGTCCG 20 ssDNA 79 MDH-qPCR-R TGCTCTTCCCCTTTGTTCTC 20 ssDNA 80 C. Primers used for cloning BiP2 promoter, TBF1 genomic fragment and 5′UTR of TBF1 TBF1-cDNA-GW-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGACGGCTGTG 57 ssDNA 81 ACGGCGGCGCAAAG TBF1-cDNA-GW-R(STOP) GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAGTTGCAGACTT 55 ssDNA 82 TGCTGCTTTTC TBF1-cDNA-GW-R GGGGACCACTTTGTACAAGAAAGCTGGGTCGTTGCAGACTTTG 55 ssDNA 83 (NON_STOP) CTGCTTTTCCTC TBF1-promoter-GW-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTA 60 ssDNA 84 CGACGACTAGTTTACAGAGAATTTGGAC BiP2-promoter-GW-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTATTCCGGTTCTTTT 56 ssDNA 85 CCACTCCTAATG BiP2-promoter-GW-R GGGGACCACTTTGTACAAGAAAGCTGGGTCATCGGAAACTTTT 53 ssDNA 86 GCGTACGATC TBF1 5′UTR-GW-F GGGGACAAGTTTGTACAAAAAAGCAGGCTTATTTCTTACAAAG 54 ssDNA 87 GTAGGACCAAC TBF1 5′UTR-GW-R GGGGACCACTTTGTACAAGAAAGCTGGGTCGTAAGTGTTGAGC 52 ssDNA 88 TGACGAATG uORF1 Phe->Leu-F GCTCCGGCGAAGTCTGGTCGTCGTCTTCATC 31 ssDNA 89 uORF1 Phe->Leu-R GATGAAGACGACGACCAGACTTCGCCGGAGC 31 ssDNA 90 uORF2 Phe->Leu-F GATTTTTCCTTAACTGGAAGAAACCAAACG 30 ssDNA 91 uORF2 Phe->Leu-R CGTTTGGTTTCTTCCAGTTAAGGAAAAATC 30 ssDNA 92 pTB3-dhAGT-TBF1 5′UTR-F GAGAAATTGAAGAGCGCAACGAACTACGAGCGGATCC 58 ssDNA 93 TTTCTTACAAAGGTAGGACC pTB3-dhAGT-TBF1 5′UTR-R GGACACGGCGACGATGCAGTTCAATGGTCGAACGTAAGTGTTG 55 ssDNA 94 AGCTGACGAATG D. Oligos used for EMSA TL1 oligo (EMSA) TCCAGTGCTGAAGAAGAATTCTACG 25 ssDNA 95 mTL1 oligo (EMSA) TCCAGTGTCATCACGTGTTTCTACG 25 ssDNA 96 E. tRNA Probe Sequences tRNA-Phe AGCGTGGATCGAACACGCGACCTTCAGATCTTCAGTCTGACGCT 59 ssDNA 97 CTCCCAACTGAGCTA tRNA-Asp GCCGGGGATCGAACCCGGGTCACCCGCGTGACAGGCGGGAAT 59 ssDNA 98 ACTTACCACTATACTAC F. TBF1 region Exact TL1 motif GAAGAAGAA 9 DNA 99 Degenerate TL1 motif GXXXGXXXX, approximating degenerate motif, G-(A/G)- 9 Syn- 100 (AGT)-G-(ACG)-(ACG)-(ACG)-(AC)-(ACGT) as noted in the thetic frequencies specified in the weight matrix of Table 6. Genomic TBF1 region Genomic TBF1 region from Arabidopsis thaliana including 5085 DNA 101 TBF1 promoter, and regions encoding uORF1, uORF1, and TBF1 polypeptides (in exons 1 and 2, separated by TBF1 intron) See FIGS. 19 and 20 displaying the sequence and genetic elements of the TBF1 region in compact form. Beginning with Aaaattttca . . . Ending with . . . acagaaacat tttct 5085 uORF1 aa MVVVFIFFLHHQIFP 15 AA 102 uORF2 aa MEETKRNSDLLRSRVFLSGFYCWDWEFLTALLLFSC 36 AA 103 TBF1 aa (1^(st )Exon) MTAVTAAQRSVPAPFLSKTYQLVDDHSTDDVVSWNEEGTAFVV 73 AA 104 WKTAEFAKDLLPQYFKH NNFSSFIRQLNTY TBF1 aa (2^(nd) Exon) GFRKTVPDKWEFANDYFRRGGEDLLTDIRRRKSVIASTAGKCVVVGS 211 AA 105 PSESNSGGGDDHGSSSTSSPGSSKNPGSVENMVADLSGENEKLKRENN NLSSELAAAKKQ RDELVTFLTGHLKVRPEQIDKMIKGGKFKPVESDEESECEGCDGGGGAE EGVGEGLKLFG VWLKGERKKRDRDEKNYVVSGSRMTEIKNVDFHAPLWKSSKVCN TBF1 aa (1^(st )and 2^(nd )Exons) MTAVTAAQRSVPAPFLSKTYQLVDDHSTDDVVSWNEEGTAFVVWKT 284 AA 106 AEFAKDLLPQYFKH NNFSSFIRQLNTY GFRKTVPDKWEFANDYFRRGGEDLLTDIRRRKSVIASTAGKCVVVGS PSESNSGGGDDHGSSSTSSPGSSKNPGSVENMVADLSGENEKLKRENN NLSSELAAAKKQ RDELVTFLTGHLKVRPEQIDKMIKGGKFKPVESDEESECEGCDGGGGAE EGVGEGLKLFG VWLKGERKKRDRDEKNYVVSGSRMTEIKNVDFHAPLWKSSKVCN TBF1 genomic region with The 4601-bp genomic DNA containing the TBF1 4601 DNA 107 STOP codon used for promoter and the coding region(SEQ ID NO: complementation 107) with the TBF1 intron was amplified using primers TBF1-promoter-GW-F(SEQ ID NO: 84) and TBF1-cDNA-GW-R(SEQ ID NO: 82) Beginning with cgacgactag . . . Ending with . . . tctgcaacta a 4601 TBF1 UTR through first exon The DNA fragment containing the 5′ 670 DNA 108 of TBF1 ORF untranslated region (UTR) and the first exon of TBF1 was amplified by polymerase chain reaction (PCR) using primers TBF1 5′UTR-GW-F (SEQ ID NO: 87) and TBF1 5′UTR-GW-R(SEQ ID NO: 88), designated uORF1-uORF2-TBF1 1^(st) exon Beginning with tttcttacaa . . . Ending with . . . caacacttac 670 BiP2 promoter fragment A 352-bp long fragment of the BiP2 promoter 352 DNA 109 containing four TL1 motifs was PCR-amplified using primers BiP2-promoter-GW-F(SEQ ID NO: 85) and BiP2-promoter-GW-R(SEQ ID NO: 86), cloned into the pDONR207 Gateway Entry vector and subsequently recombined into yeast one hybrid bait destination vectors pMW2 and pMW3. Beginning with Ttccggttct . . . Ending with . . . agtttccgat Genomic TBF1 region The 4,598-bp genomic DNA containing the TBF1 4598 DNA 110 NON_STOP promoter and the coding region(SEQ ID NO: 107) was amplified using primers TBF1- promoter-GW-F(SEQ ID NO: 84) and TBF1- cDNA-GW-R(NON_STOP)(SEQ ID NO: 83). The resulting fragment was cloned into pDONR207, subsequently recombined into pMDC107 and used to generate a genomic TBF1-GFP fusion. Beginning with cgacgactag . . . Ending with . . . AGTCTGCAAC uORF1 nucleotide seq Nucleotide sequence encoding Arabidopsis 48 DNA 111 thaliana upstream ORF1(uORF1) polypeptide ATGGTCGTCGTCTTCATCTTCTTCCTCCATCATCAG ATTTTTCCTTAA uORF2 nucleotide seq Nucleotide sequence encoding Arabidopsis 111 DNA 112 thaliana upstream ORF2(uORF2) polypeptide ATGGAAGAAACCAAACGAAACTCCGATCTTCTCCG TTCTCGTGTTTTCCTCTCTGGCTTTTATTGCTGGGAT TGGGAATTTCTCACCGCTCTCTTGCTTTTTAGTTGC TGA TBF1 promoter Arabidopsis thaliana TBF1 promoter region 3354 DNA 113 Beginning with cgacgactag . . . Ending with . . . ttcgccggag TBF1 genomic seq Coding sequence only for the Arabidopsis 1047 DNA 114 thaliana TBF1 open reading frame with exon 1, intron, and exon 2, starting with an ATG codon and terminating with a TAA stop codon. Beginning with ATGACGGCTG . . . Ending with . . . CTGCAACTAA

TABLE 2 Host strains Designation Organism Relevant Genotype Reference/Source Psm ES4326 Pseudomonas syringae ES4326 [60] pv. maculicola (Psm) Psm ES4326/ Pseudomonas syringae ES4326 avrRpt2 [82] avrRpt2 pv. maculicola (Psm) carrying an avirulent effector avrRpt2 YM4271 Saccharomyces Yeast strain used in yeast-one- [71 ] cerevisiae hybrid experiments, MATa, ura3- Clontech 52, his3-200, ade2-101, ade5, lys2- 801, leu2-3, 112, trpl-901, tyr1- 501, ga14D, ga18D, ade5::hisG BY4742 Saccharomyces yeast strain BY4742 MATαhis3Δ1 [72] cerevisiae leu2Δ0 lys2Δ0 ura3Δ0 pep4Δ::KAN^(R) International Yeast Deletion Consortium Strain 1 Saccharomyces Strain 1 derived from YM4271 that This study cerevisiae has the WT BiP2 promoter driving both HIS3 and URA3 genes. Strain 2 Saccharomyces Strain 2 derived from YM4271 that This study cerevisiae has the WT BiP2 promoter for HIS3 and the mutated BiP2 promoter [61]for URA3 (Figure 2). GV3101 Agrobacterium Bacterial strain for floral-dip based [73] tumefaciens plant transformation, rifampicin- resistant

TABLE 3 Plasmids Designation Markers Description Reference/Source pMDC140 Kan^(R), Gateway plant expression vector carrying a [56] Hygro^(R) Gateway cassette cloned downstream of the 35S promoter and upstream of the GUS reporter gene; vector confers kanamycin resistance in E. coli and hygromycin resistance in transgenic plants. pMDC140- Kan^(R), WT region uORF1-uORF2 Hygro^(R) pMDC140- Kan^(R), One A-to-C point mutation was introduced This study mORF1- Hygro^(R) into the start codons (ATG) of uORF1. The uORF2 mutant mORF1-uORF2 sequence was inserted downstream of the constitutive 35S promoter and upstream of the coding region of the GUS reporter in pMDC140 through recombination [56]. pMDC140- Kan^(R), One A-to-C point mutation was introduced This study uORF1- Hygro^(R) into the start codons (ATG) of uORF2. The mORF2 mutant uORF1-mORF2 sequence was inserted downstream of the constitutive 35S promoter and upstream of the coding region of the GUS reporter in pMDC140 through recombination [56]. pMDC140- Kan^(R), Two A-to-C point mutations were This study mORF1- Hygro^(R) introduced into the start codons (ATG) of mORF2 uORF1 and uORF2. The mutant mORF1- mORF2 sequences were inserted downstream of the constitutive 35S promoter and upstream of the coding region of the GUS reporter in pMDC140 through recombination [56]. pTB3 Plasmid comprising S. cerevisiae DHFR Chandra Tucker promoter and reporter gene pTB3-unstable MTXR^(R) DHFR reporter gene carried by plasmid This study DHFR&MTX pTB3 was engineered to make an unstable resistance enzyme [37] and to contain the L22F/F31S mutations that confer resistance to L22F/F31S methotrexate (MTX) [58]. pDONR207 Gent^(R), Gateway Entry vector Invitrogen Cat^(R) pMDC123 Kan^(R), Gateway plant expression vector that [56] Cat^(R), confers kanamycin and chloramphenicol HphR resistance in E. coli and hygromycin resistance in transgenic plants. pMW2 Amp^(R), Yeast one hybrid destination vector, HIS3 [83] Cat^(R), reporter HIS3 pMW3 Amp ^(R), Yeast one hybrid destination vector, LacZ [83] Cat^(R), reporter URA3 pENTR207- Gent^(R) A 352-bp long fragment of the BiP2 This study BiP2 promoter cloned into the pDONR207 Gateway Entry vector pMW2- BiP2 Amp^(R), pMW2 plasmid containing a 352-bp long This study HIS3 fragment of the BiP2 promoter pMW3- BiP2 Amp^(R), pMW3 plasmid containing a 352-bp long This study URA3 fragment of the BiP2 promoter pMDC123 Kan^(R), To perform genetic complementation of This study TBF1p:TBF1 tbf1, the 4,601-bp genomic DNA containing the TBF1 promoter and the coding region (SEQ ID NO: 107) was amplified using primers TBF1-promoter-GW-F (SEQ ID NO: 84) and TBF1-cDNA-GW-R (SEQ ID NO: 82) cloned into the vector pDONR207 using the Gateway technology (Invitrogen), and inserted by recombination into the destination vector pMDC123 [6]. pMDC107 Kan^(R), Gateway plant expression vector carrying a [56] Hygro^(R) Gateway cassette cloned upstream of the GFP reporter gene; vector confers kanamycin resistance in E. coli and hygromycin resistance in transgenic plants. pMDC107 Kan^(R), The genomic TBF1 to GFP fusion was also This study TBF1p:TBF1- Hygro^(R) generated by recombining pENTR207 GFP TBF1p:TBF1 NON_STOP (amplified using primers TBF1-promoter-GW-F (SEQ ID NO: 84) and TBF1-cDNA-GW-R (NON_STOP) (SEQ ID NO: 83) into the destination vector pMDC107 [66]. The resulting destination clone pMDC107 TBF1p:TBF1-GFP, was transformed into Agrobacterium tumefaciens strain GV3101 and introduced into tbf1 mutant plants. A homozygous T3 line was selected for additional analysis.

Table 4 Description and sources of cloned genes GenBank Accession Designation Full Name Origin Number Reference/Source TBF1 4,601-bp genomic DNA Arabidopsis At4g36990/ Arabidopsis genomic DNA containing the TBF1 thaliana NM _119862 (gDNA), This study promoter and the coding region GUS (uidA) 1809 by long gene Escherichia coli NP_ 416134 [75][76][76] encoding beta- Invitrogen glucuronidase LacZ 3072 by long gene Escherichia coli AB199820 [78][79][80] encoding beta- Invitrogen galactosidase GFP 714 by long gene encoding Aequorea AAA27722 [81] green fluorescent protein victoria Invitrogen BiP2 promoter Arabidopsis At4g42020/ Arabidopsis genomic DNA fragment thaliana NM_123567 (gDNA), This study

TABLE 5 Features of Engineered Cell Lines or Plants Cell line Description Reference/Source Strain 1/ pMW2 BiP2 WT, pMW3 BiP2 WT, pDESTAD-TBF1 This study pDESTAD-TBF1 Strain 1/ pMW2 BiP2 WT, pMW3 BiP2 WT, pDEST-AD This study pDEST-AD Strain 2/ pMW2 BiP2 WT, pMW3 mBiP2, pDESTAD-TBF1 This study pDESTAD-TBF1 Strain 2/ pMW2 BiP2 WT, pMW3 mBiP2, pDEST-AD This study pDESTAD-AD GV3101/ The resulting destination clone pMDC123 pMDC123 TBF1p:TBF1 TBF1p:TBF1, was transformed into Agrobacterium tumefaciens strain GV3101. Constructs were This study introduced into tbf1 mutant plants by the floral dipping method [67]. GV3101/ The genomic TBF1 to GFP fusion was generated by This study pMDC107 TBF1p:TBF1-GFP recombining pENTR207 TBF1p:TBF1 into the destination vector pMDC107 [66]. The resulting destination clone pMDC107 TBF1p:TBF1-GFP, was transformed into Agrobacterium tumefaciens strain GV3101 and introduced into tbf1 mutant plants. A homozygous T3 line was selected for additional analysis. GV3101/ Transiently expressed in Nicotiana benthamiana This study pMDC140-uORF1-uORF2 using Agrobacterium tumefaciens GV3101/ Transiently expressed in Nicotiana benthamiana This study pMDC140-uorf1-uORF2 using Agrobacterium tumefaciens GV3101/ Transiently expressed in Nicotiana benthamiana This study pMDC140-uORF1-uorf2 using Agrobacterium tumefaciens GV3101/ Transiently expressed in Nicotiana benthamiana This study pMDC140-uorf1-uorf2 using Agrobacterium tumefaciens GV3101/ Constructs were transformed into Arabidopsis wild- This study pMDC140-uORF1-uORF2 type Col-0 plants. Two stable transgenic T3 lines homozygous for each construct were chosen for quantitative GUS assay [3] at 0, 0.5, 1, 2, 3, 4 and 8 hours after Psm ES4326/avrRpt2 infiltration (OD_(600nm) = 0.02). GV3101/ Same as above except that the mutant construct This study pMDC140-uorf1-uorf2 was used here (wild-type construct above) Arabidopsis plants Control genotype ABRC* (Columbia-0) Arabidopsis plants SALK 104713 ABRC* (tbf1 mutant) Arabidopsis plants Homozygous T₃ Arabidopsis line used for This study (tbf1 background) complementation assays pMDC123 TBF1p:TBF1 Arabidopsis plants Homozygous T₃ Arabidopsis line used for ChIP This study (tbf1 background) experiments pMDC107 TBF1p:TBF1-GFP *Arabidopsis Biological Resource Center (ABRC, abrc.osu.edu).

Experimental Procedures Translational Analysis of uORF1-uORF2-GUS

The DNA fragment containing the 5′ untranslated region (UTR) and the first exon of TBF1 (designated uORF1-uORF2-TBF1 1^(st) exon) was amplified by polymerase chain reaction (PCR) using primers TBF1 5′UTR-GW-F (SEQ ID NO: 87) and TBF1 5′UTR-GW-R (SEQ ID NO: 88), wherein the DNA fragment is represented by the nucleic acid set forth as SEQ ID NO: 108 and cloned into the Gateway vector pDONR207 (Invitrogen). Two A-to-C point mutations were introduced, either separately or together, into the start codons (ATG) of uORF1 and uORF2. The WT and mutant uORF1-uORF2 sequences were inserted downstream of the constitutive 35S promoter and upstream of the coding region of the GUS reporter in pMDC140 through recombination [56]. The resulting translational reporter plasmids (designated pMDC140-uORF1-uORF2-GUS and its mutant variants pMDC140-uorf1-uORF2-GUS, pMDC140-uORF1-uorf2-GUS and pMDC140-uorf1-uorf2-GUS (Table E3) were transformed into Col-0 WT plants or transiently-expressed in Nicotiana benthamiana using Agrobacterium tumefaciens [57]. For Arabidopsis stable transgenic lines, two independent T₃ lines homozygous for each construct (as set forth in Table 5) were chosen for quantitative GUS assay [3] at 0, 0.5, 1, 2, 3, 4 and 8 hours after Psm ES4326/avrRpt2 infiltration (OD_(600nm)=0.02).

Yeast Growth Assay Using the DHFR Reporter

The DHFR reporter gene carried by plasmid pTB3 was engineered to make an unstable enzyme [37] and to contain the L22F/F31S mutations that confer resistance to methotrexate (MTX) [58]. The uORF1-uORF2 of TBF1 was translationally-fused to the coding region of the DHFR reporter and integrated into the genome of yeast strain BY4742 by homologous recombination. Equal amounts of yeast culture grown in liquid media (SD-Leu) were inoculated into SD-Leu-Phe double drop-out media supplemented with 15 mg/L or 75 mg/L phenylalanine. In other experiments, yeast cultures were also grown in Phe-rich, Asp-deficient media supplemented with 15 mM tobramycin (TOB) (Sigma, St. Louis, Mich., USA), a known inhibitor of yeast tRNA^(Asp) aspartylation. MTX was added to all cultures at the final concentration of 80 μM to inhibit the endogenous DHFR activity. Yeast growth, which was dependent on the expression of the recombinant DHFR reporter in the presence of MTX, was measured using optical density (OD_(600nm)) over a 32-hour time course.

Genome-Wide Search of the TL1 cis-Element

To perform a genome-wide search for the TL1 cis-element, 1000-bp upstream sequences with a cutoff at the adjacent gene were fetched from the Arabidopsis Information Resource website (arabidopsis.org) and analyzed using the Athena website software (bioinformatics2.wsu.edu). The sequence GAAGAAGAA (SEQ ID NO: 99) was considered as the exact TL1 motif. Degeneracy of the TL1 element was based on Wang et al. [61] and shown in Table 6, below. To control the level of degeneracy, the total weight of the hit was restricted to be more than 664. The exact (SEQ ID NO: 99) and degenerate TL1 motifs (approximately represented by SEQ ID NO: 100) were searched for using the scan_for_matches software, available at (iubio.bio.indiana.edu).

TABLE 6 Weight matrix for degenerate TL1 element used in the genome-wide search for TL1 Motif G A A G A A G A A Position 1 2 3 4 5 6 7 8 9 A 0 88 85 0 91 91 12 62 65 C 0 0 0 0 3 6 6 38 26 G 100 12 3 100 6 3 82 0 6 T 0 0 12 0 0 0 0 0 3

Yeast One Hybrid Assays

Y1H assays were performed according to a previously-published protocol [62]. In brief, a 352-bp long fragment of the BiP2 promoter (SEQ ID NO: 109) was cloned into the pDONR207 Gateway Entry vector, recombined into pMW2 and pMW3 vectors, and then integrated into the HIS3 and URA3 loci in yeast, respectively. Strain 1 has the WT BiP2 promoter driving both HIS3 and URA3 genes, and Strain 2 has the WT BiP2 promoter for HIS3 and the mutated BiP2 promoter [61] for URA3 (Table 2, FIG. 2). Both strains were transformed with the construct pDESTAD-TBF1 or pDEST-AD, and the resulting yeast colonies were pooled and spotted on selection media (SD-Leu-Trp-Ura) (Clontech) supplemented with increasing concentrations of 3-AT (Sigma). Yeast growth was recorded 3 days later.

Quantitative β-Galactosidase Assay

The assay was modified from protocols described in earlier studies [63]. In brief, 0.1 ml of yeast transformant extract was added to 0.9 ml of Z buffer and warmed to 28° C. Reactions were initiated by addition of 0.2 ml of ONPG substrate (4 mg/ml) in Z buffer and terminated with 0.5 ml of 1 M Na₂CO₃. Reactions were terminated within the linear range of the assay (OD_(420nm)<1.0). The β-galactosidase activity in yeast supernatants was normalized to their protein concentrations. The data represent the average from three dilutions of extracts.

Electrophoretic Mobility Shift Assay

The assay was performed as described in earlier studies [61]. Briefly, 3-week-old plants were treated with 1 mM SA for 4 hrs before leaf tissues were harvested. 40,000 cpm of labeled probe was added to 10 μg of protein, incubated in a buffer containing 12 mM HEPES pH 8.0, 60 mM KCl, 2 mM MgCl₂, 0.1 mM EDTA, 12% glycerol, and 0.3 mM DTT for 20 min, and separated on a 5% polyacrylamide gel. DNA-protein interactions were detected using autoradiography.

Chromatin Immunoprecipitation (ChIP)

ChIP assays were performed as described in earlier studies [64]. For each sample, 1 g of leaves from 3-week-old Arabidopsis plants was crosslinked with 1% formaldehyde under vacuum for 15 min, followed by addition of glycine to a final concentration of 0.1 M. The leaves were washed with water and then ground in liquid nitrogen. Arabidopsis nuclei were isolated and sonicated in Bioruptor® sonicator (Diagenode). The TBF1-GFP-tagged protein was immunoprecipitated using 1 μl of an anti-GFP antibody ab-290 (Abcam) that was first coupled to the protein G Dynabeads (Invitrogen). The purified ChIP DNA samples were subject to real-time PCR analysis. The amount of each amplicon was normalized to the input. The relative amount (fold-enrichment) of each signal was determined by the ratio of normalized ChIP signals between samples. The primer sequences used for ChIP analysis are listed in Table 1}.

qRT-PCR

Total RNA was extracted from 3-week-old plants with and without 1 mM SA treatment at different time points. RNA extractions were performed using TRIzol Reagent (Ambion). RNA samples were reverse-transcribed into cDNA using SuperScript III Reverse Transcriptase (Invitrogen). The cDNA was quantified using gene specific primers (Table 1, above) and the POWER SYBR GREEN PCR Master Mix (Applied Biosystems) in a LightCycler (Roche) or RealPlex S (Eppendorf).

Microarray

Arabidopsis plants (Columbia-0 and tbf1 mutant) were grown on soil (Metro Mix 360) at 22° C. under a 16/8 hr light/dark cycle for 3 weeks and treated with 1 mM SA for 6 hrs (spray) or 10 μM elf18 for 2 hrs (infiltration into leaves). Mock treatments with water were included for both spray and infiltration. The RNA, extracted with TRIzol (Ambion) and labeled with MessageAmp Premier RNA Amplification Kit (Ambion), was hybridized with GeneChip Arabidopsis ATH1Genome Array (Affymetrix), and subsequently washed and scanned at the Duke Microarray Facility. Experiments were repeated three times using independently-grown and treated plants. The resulting data were normalized using Gene-Spring GX Software (RMA algorithm; Agilent). Two-way ANOVA with the Benjamini-Hochberg multiple comparison correction was used to identify TBF1-dependent genes (i.e., with significant interaction between genotypes and treatments, p-value<0.05). The SA- and elf18-responsive genes (fold change>2) were found through unpaired Student's t test with the Benjamini-Hochberg multiple comparison correction (p-value<0.05). The Venn diagram was adapted from Venny [5]. To generate the heatmaps of SA- or elf18-upregulated and down-regulated genes, −log10p-values of induced genes and log10p-values of repressed genes from Student's t test were used. Higher positive values represent greater induction, and lower negative values indicate greater repression. For TBF1 dependence, −log10p-values from two-way ANOVA tests were used. The gene ontology analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) (available at david.abcc.ncifcrf.gov/).

Tunicamycin Sensitivity Assay

This assay was performed as described in earlier studies [61].

Genetic Complementation of tbf1

To perform genetic complementation of tbf1, the 4,601-bp genomic DNA containing the TBF1 promoter and the coding region (SEQ ID NO: 107) was amplified using primers TBF1-promoter-GW-F (SEQ ID NO: 84) and TBF1-cDNA-GW-R (SEQ ID NO: 82) (Table 1), cloned into the vector pDONR207 using the Gateway technology (Invitrogen), and inserted by recombination into the destination vector pMDC123 [6]. The resulting destination clone pMDC123 TBF1p:TBF1, was transformed into Agrobacterium tumefaciens strain GV3101. Constructs were introduced into tbf1 mutant plants by the floral dipping method [67]. T₃ transgenic plants homozygous for the transgene were further analyzed. The genomic TBF1 to GFP fusion was also generated by recombining pENTR207 TBF1p:TBF1 into the destination vector pMDC107 [66]. The resulting destination clone pMDC107 TBF1p:TBF1-GFP, was transformed into Agrobacterium tumefaciens strain GV3101 and introduced into tbf1 mutant plants. A homozygous T₃ line was selected for additional analysis.

Bacterial Infection Assay

Infection of Arabidopsis plants with Pseudomonas syringae pv. maculicola (Psm) ES4326 was performed as described previously [68]. To test for enhanced disease susceptibility, a bacterial suspension at OD_(600nm)=0.0001 was infiltrated into 2-3 leaves per plant and 12 plants/genotype. Bacterial growth was quantified 3 days later. To test for SAR and MAMP-induced resistance, plants were pre-treated with relevant compounds (1 mM SA, spray 24 hrs prior to infection; 10 μM elf18, infiltration 2 hrs prior to infection; 10 μM flg22, infiltration 2 hrs prior to infection), and subsequently inoculated with Psm ES4326 (OD_(600nm)=0.001) into 2-3 leaves per plant and 12 plants/genotype/treatment. Sampling was performed 3 days post inoculation.

PR1 Protein Secretion

Three-week-old plants were treated with 1 mM SA for 24 hrs before infiltration under vacuum in a 20 mM phosphate buffer (KH₂PO₄ and K₂HPO₄, pH=7.4). Intercellular wash fluid was collected from equal amounts of tissue by centrifuging the infiltrated leaf samples, which were packed in a syringe, for 5 min at 1500 g. As a control, total protein was also extracted from 50 mg of leaf tissue (from 3-4 independent plants) using a buffer described previously [61]. Secreted and total protein were run on 15% SDS-PAGE gels, transferred to a nitrocellulose membrane, and probed with a polyclonal rabbit antibody raised against a synthetic peptide matching the carboxy terminus of the Arabidopsis PR1 protein (1:4000 dilution, 2 hrs) followed by a goat anti-rabbit secondary antibody (Santa Cruz Biotechnology) (1:2000 dilution, 1.5 hrs). To confirm equal loading of total protein, an anti-α-tubulin antibody (Sigma) was used subsequently to probe the total protein concentration on the blot.

Western Blotting

The anti-BiP Western blotting experiment was performed as described previously [61], using leaf tissue sprayed with 1 mM SA 6 hrs prior to collection. The primary antibody was α-BiP (Santa Cruz Biotechnology, aC-19, 1:4000 dilution, overnight at 4° C.), followed by the secondary antibody (bovine anti-goat, Santa Cruz Biotechnology, 1:2000 dilution, 2 hrs, RT). The anti-phospho eIF2α Western blotting experiment was performed as described previously [69], using leaf tissue infected with Psm ES4326 expressing avrRpt2 (OD_(600nm)=0.02) over the indicated time periods. The protein extraction was carried out in presence of a phosphatase inhibitor PhosSTOP (Roche), Protease Inhibitor Cocktail (Sigma Aldrich) and proteasome inhibitor MG-115 (Sigma Aldrich). The primary antibody was α-p-eIF2α (pS51) (Epitomics, Burlingame, Calif., 1090-1), (1:1000 dilution, overnight at 4° C.), followed by the secondary antibody (goat anti-rabbit, Bio-Rad, 1:4000 dilution, 1 hr, RT).

Rapid Amplification of cDNA Ends (RACE)-PCR

RACE-PCR analyses were performed as described in manufacturer's protocol (SMART™ RACE cDNA Amplification Kit, Clontech, Mountain View, Calif., USA).

tRNA Analysis

Total RNA was extracted using TRIzol reagent (Invitrogen) according to the instructions provided by the manufacturer. Total RNA was then dissolved in 0.1 M sodium acetate (pH 5.0). mRNA was precipitated using 2 M LiCl overnight. 2 volumes of isopropanol were added to the supernatant to precipitate the tRNA. After washing with 100% ethanol, the tRNA was dissolved in 0.1 M sodium acetate. 1 μg tRNA was separated by acid urea PAGE, and transferred to NEF 976 GeneScreen Plus Hybridization Transfer Membrane (PerkinElmer) according to procedures established in earlier studies [70].

Specific tRNA species were detected by hybridization using digoxigenin-labeled DNA probes (shown in Table 1} as tRNA^(Phe) represented by SEQ ID NO: 97, and tRNA^(Asp) represented by SEQ ID NO: 98) according to the manufacturer's instructions (DIG High Prime DNA Labeling and Detection Starter Kit II, Roche Applied Science). The signal was visualized using a low-light CCD camera.

Polysome Profiling

Before extraction, a spike-in control was added into the pulverized leaf tissue at a concentration of 10⁷ copies of Alien qRT-PCR Inhibitor Alert (Agilent Technologies, USA) per mg of fresh weight. 500 mg of pulverized leaf tissue was hydrated on ice for 10 min with occasional vortexing in 3 ml of extraction buffer, containing 0.2 M Tris (pH=9.0), 0.2 M KCl, 0.025 M EGTA, 0.035 M MgCl₂, 1% (w/v) Brij-35, 1% (v/v) Triton X-100, 1% (v/v) Igepal CA 630, 1% (v/v) Tween 20, 1% (w/v) sodium deoxycholate, 1% (v/v) polyoxyethylene 10 tridecyl ether, 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 50 μg/mL cycloheximide, 50 μg/mL chloramphenicol. The hydrated tissue was centrifuged at 16,000 g for 15 min. The supernatant was then separated in a 10 ml continuous (15-60% w/v) sucrose gradient containing 400 mM potassium acetate, 25 mM potassium HEPES (pH=7.2), 15 mM magnesium acetate, 200 μM cycloheximide by ultracentrifugation at 35,000 rpm using SW 41Ti rotor (Beckman Coulter, Germany) for 10 hrs at 4° C. The gradients were fractionated into 36 fractions of about 330 μL each using automated Density Gradient Fractionation System (Teledyne Isco Inc., USA) with a simultaneous A_(254nm) trace. Total RNA was extracted from the fractions containing ribosomes using TRIzol reagent (Invitrogen) according to instructions provided by the manufacturer. mRNA was further precipitated using 2 M LiCl overnight. cDNA was prepared and qRT-PCR analyses performed as described above.

Statistical Analyses

For gene expression data, expression values were used for linear models. Effects of genotype, treatment, time, biological replicate and interactions between genotype and time, and genotype and treatment were included in the linear model where appropriate. For bacterial infection data, logarithmic transformed colony forming units (cfu) were used for linear models. Effects of genotype, treatment, time, biological replicate and interactions between genotype and time, and genotype and treatment were included in the linear model where appropriate. Bonferroni post-tests were applied to address the significant difference at individual time points between WT and mutant samples. All statistical analyses were performed using R software programs (CRAN).

Table S1 summarizes the complete list of TBF1-dependent SA- and elf18-regulated genes which are set forth supplementary data tables extracted from eight worksheets in an Excel file, herein specifically incorporated by reference, as noted above in the section entitled “Incorporation-By-Reference Under 37 CFR 1.58 to Large Tables Including Supplemental Tables of Information Included In Earlier Priority Applications”.

TABLE S1 Tables SIA-SIH, incorporated by reference, listing TBFI-dependent SA- and elf18-regulated genes Excel Table # Sheet # Description SA elf18 #Data Rows S1A 1 SA induced only + 528 S1B 2 SA repressed only — 534 S1C 3 elf18 induced only + 477 S1D 4 elf18 repressed only — 1098 S1E 5 SA and elf18 induced + + 37 S1F 6 SA and elf18 repressed — — 110 S1G 7 SA induced elf18 repressed + — 60 S1H 8 SA repressed elf18 induced — + 22

Example 1 Results TBF1 is a TF that Binds to the TL1 cis-Element Enriched in Defense-Related Gene Promoters

The TL1 cis-element (consensus sequence GAAGAAGAA) in the ER-resident genes is essential for their activation in response to SA induction [3]. To determine whether this cis element is important only for the ER-resident genes or also for induction of other defense-related functions, we examined the promoter regions (1000 by upstream of the ATG start codon) of genes regulated by the SA analog BTH (benzothiadiazole) (available at affy.arabidopsis.info/narrays/experimentbrowse.pl, experiment ID:NASCARRAYS-392) [11] and by the MAMP signals flg22 and elf26 (the first 26 amino acids of EF-Tu) (available at www.ebi.ac.uk/arrayexpress/, experiment ID: E-MEXP-547) [12] using the Athena program (www.bioinformatics2.wsu.edu/Athena). We found that the TL1 cis-element is enriched in the promoter regions of genes regulated by elf26 (p-value<0.001) and flg22 (p-value<0.01), indicating that this novel element may play a role in MTI. No significant enrichment of TL1 was detected when all of the BTH-affected promoters were analyzed, even though the element was first discovered in the SA-induced, NPR1-dependent ER-resident genes [3].

To search for the TF that regulates the TL1 cis-element (i.e., TBF1), we submitted the TL1 core sequence GAAGAAGAA to the TFSEARCH database (www.cbrc.jp/research/db/TFSEARCH.html) and found several HSFs of Saccharomyces cerevisiae and Drosophila melanogaster as potential candidates. The Arabidopsis genome contains 21 HSF-like genes. Several reports have indicated the involvement of the HSFs in immediate heat response, acquired thermotolerance, sensing of reactive oxygen species (ROS), and seed development [13, 14]. To identify a candidate gene for TBF1, we first examined the expression profiles of the Arabidopsis HSF family members using available microarray data in response to BTH induction (affy.arabidopsis.info/narrays/experimentbrowse.pl, experiment ID: NASCARRAYS-392) [11] and to the virulent and avirulent Pseudomonas syringae pv. maculicola (Psm) ES4326 bacteria (affy.arabidopsis.info/narrays/experimentbrowse.pl, experiment ID: NASCARRAYS-168). Only one gene family member, HSF4 (also known as HsfB1; AT4G36990), was strongly induced by these treatments. Because Arabidopsis HSF4 and its tomato homolog do not functionally complement the yeast hsf1 mutant strain [15] (Daniel Neef and Dennis Thiele, personal communication), and its overexpression has little effect on heat shock protein expression or thermotolerance [16, 17], we thought that HSF4 does not encode a typical heat shock factor. Its pathogen-inducible expression pattern suggests that it has a novel function related to plant immunity, and is a candidate for TBF1.

We carried out additional studies to demonstrate that HSF4 is the TL1 cis-element TBF1 involving a yeast one-hybrid (Y1H) vector system, in which the promoter fragment of BiP2, containing multiple functional TL1 cis-elements, was used as bait [3]. Two yeast bait strains containing the WT and the mutant (mTL1) BiP2 promoters, respectively, were constructed (FIG. 2). Expression of TBF1-AD (containing the activation domain) in Strain 1, activated both HIS3 and LacZ reporters driven by the WT BiP2 promoter (FIGS. 1A and 1B). The binding specificity of TBF1 to TL1 was confirmed in Strain 2, where the two single-nucleotide substitutions in the mTL1 core binding sequence blocked the induction of LacZ, while the control HIS3 reporter with the WT TL1 was induced normally (FIG. 1B).

TBF1 binding to the TL1 cis-element was further demonstrated using electrophoretic mobility shift assays with protein extracts from both WT and an insertional knock-out TBF1 mutant, tbf1 (FIG. 3). As shown in FIG. 1C, WT displayed an up-shifted band, whose intensity was further enhanced in the extract made from plants treated with SA. This band was diminished in the tbf1 mutant extract, indicating that TBF1 is required for the DNA-protein complex formation. Competition assays, using non-radioactive TL1 and mutant mTL1 probes, indicated that the observed TBF1 binding was specific to the TL1 consensus sequence.

To test TBF1 DNA-binding activity in vivo, we generated transgenic tbf1 plants expressing a translational fusion between TBF1 and GFP driven by the endogenous TBF1 promoter (TBF1p:TBF1-GFP). Because the fusion protein was proven to be biologically-active through genetic complementation of the tbf1 mutant phenotype (FIG. 4), we used it for chromatin immunoprecipitation (ChIP). As shown in FIG. 1D, using six pairs of primers spanning different regions of the BiP2 promoter (SEQ ID NOS: 1-12), we detected sequence enrichment corresponding to the TL1-containing region 2 in both uninduced and SA-treated samples, region 3 in the SA-treated sample, and region 4 in the uninduced sample. No enrichment was found in regions 1, 5 and 6 that do not contain the TL1 element.

TBF1 is a Major Molecular Switch Involved in Transcriptional Reprogramming Induced by SA and elf18

In earlier studies, we showed that the TL1 cis-element is present in many ER-resident genes [3]. In this study, we tested if SA-mediated induction of these genes is dependent on TBF1. We found that the induction of BiP2 and CRT3, containing multiple copies of TL1 elements in their promoters, was compromised in the tbf1 mutant and in npr1-1 (FIG. 5A). BiP2 protein accumulation was not induced in the SA-treated tbf1 mutant plants (FIG. 6). In contrast to BiP2 and CRT3, induction of two other ER-resident genes, BiP3 and CRT1, which do not have TL1 in their promoters, was not affected in tbf1 (FIG. 5B), confirming the specificity of TBF1 to TL1.

Enrichment of the TL1 cis-element in immune-induced ER-resident gene promoters, as well as promoters responsive to diverse immune signals [3, 18, 19], prompted us to perform a genome-wide transcriptional profiling experiment to determine the global effect of TBF1. WT and tbf1 plants challenged with SA for 6 hours or elf18 for 2 hours, were used to generate probes for the Affymetrix ATH1 GeneChip (Affymetrix, Santa Clara, Calif.). We noted that 1269 and 1792 TBF1-dependent genes were differentially-regulated by SA and elf18, respectively (fold change>2, p-values<0.05), but only a small number of genes (˜8%) were regulated by both signals (FIG. 5C, Table S1, incorporated as a large table by reference), indicating that TBF1 controls distinct output genes in SAR and MTI. The total numbers of significantly-induced and repressed genes (the top heatmaps in FIGS. 5D and 5E), the degrees of TBF1 dependency (the middle heatmaps), and the numbers of TL1 cis-elements present in the gene promoters (the bottom heatmaps) indicate that TBF1 plays a greater role in SA- and elf18-mediated transcription repression, than in induction. These results are in agreement with earlier studies indicating that class B-Hsfs mainly act as repressors of target gene expression [20, 21].

To identify the biological functions induced and repressed by TBF1, we performed gene ontology (GO) analyses and selected functional categories that were significant at p≤0.001. We identified a significantly-enriched cluster of SA-induced secretory pathway genes (FIG. 5D “membrane proteins”, Table S1, incorporated as a large table by reference), which were in agreement with our earlier findings [3]. We also identified several major functional categories comprising genes known to encode defense-related proteins (FIGS. 5D and 5E), such as the master regulator of SA pathway NPR1, a TGA-class TF, several WRKY family members, EDSS, ADR1, metacaspase 2, membrane-associated proteins like SNAP33 and members of the MLO family, and several kinases including RLKs, MAPKKs and WAKs (Table S1) [22]. These genes appear to be directly controlled by TBF1, since their regulatory regions contain the TL1 element (Table S1). A strong enrichment of ribosomal proteins was also noted among the elf18-induced TBF1-dependent genes, suggesting that significant translation events occur after MAMP induction.

Upon SA treatment, TBF1 down-regulates genes encoding chloroplast proteins (FIG. 5D), an effect that is known to be associated with SA [23]. Chloroplast function-related genes were even more profoundly repressed by elf18 (FIG. 5E). These genes encode several structural and regulatory proteins of the chloroplast (e.g., PsbR subunit of photosystem II, chloroplast o-succinylbenzoyl-CoA ligase, plastid ribosomal protein, a subunit of the chloroplast NAD(P)H dehydrogenase complex and components involved in thylakoid membrane biogenesis) (Table S1). Loss-of-function mutants involving these genes display a variety of developmental defects, such as decreased photosynthesis rate, reduced chloroplast number, pale pigmentation, dwarfism and lethality [24]. These metabolic and chloroplast-related genes are likely direct targets of TBF1, since their regulatory regions contain the TL1 element. Taken together, these results suggest that TBF1 is used in plants to rapidly reprogram cellular transcription after an infection, diverting energy resources from growth and development functions to cope with pathogenic responses.

The GO analyses indicated that elf18 treatment had a significant inhibitory effect on both abiotic stress and defense responses through TBF1, which was unexpected (FIG. 5E). These repressed genes are involved in jasmonate (JA), ethylene and auxin biosynthesis or signaling pathways (e.g., lipoxygenases, JAZ2, WRKY33, CEV1, and SAUR and IAA families) (Table S1), which are known to be down-regulated during SA-mediated defense [25-27].

The expression changes observed in the microarray experiment were also confirmed through qRT-PCR experiments of independent biological samples on 26 selected genes representing several GO categories, which are illustrated in FIGS. 5D, 5E, and FIGS. 7-10.

TBF1 Plays a Key Role in the Growth-to-Defense Transition

To determine if TBF1 is a major molecular switch involved in the transition from growth to defense functions, we first measured the growth of both WT and the tbf1 mutant plants. As shown in FIG. 11A, in the absence of SA or elf18, the tbf1 mutant grew at a similar rate as the WT plant. In the presence of elf18 or increasing concentrations of SA, however, growth of the WT plants was significantly inhibited. The inhibitory effect was partially-alleviated in the tbf1 mutant. In contrast, another MAMP signal, flg22, exerted a similar growth-suppressing effect on WT and tbf1 seedlings (FIG. 12).

We also performed a series of tests to determine the stress responses mediated by TBF1. Although the tbf1 mutant has been shown to have a normal heat-induced marker gene expression profile (FIG. 13), and plays no detectable role in the heat shock response [28], its defect in the induction of multiple chaperone genes prompted us to examine the unfolded protein response (UPR). In mutant plants treated with the UPR inducer tunicamycin, seedling survival rate for tbf1 was only ˜20% compared to ˜60% for WT (FIG. 11B), indicating that TBF1 plays a role in UPR. In earlier studies, SA-inducible ER-resident genes are required for efficient secretion of antimicrobial PR proteins [3]. Because TBF1 was shown to control SA-mediated induction of these genes (FIGS. 5A and 5D, Table S1, incorporated as a large table by reference), we tested the secretion of PR1 in the tbf1 plants. While the PR1 transcript induction and total PR1 protein levels were unchanged in tbf1 (FIG. 11C and FIG. 14), secreted PR1 in the intercellular wash fluid was dramatically reduced in the tbf1 mutant, compared to the WT sample. This phenotype was complemented in the transgenic line carrying a genomic fragment comprising upstream regulatory sequences (3,554 bp) (SEQ ID NO: 113) and the coding region (1,047 bp) of TBF1 (TBF1 Compl.) (SEQ ID NO: 114). The bip2 dad1 double mutant, which was previously shown to be defective in PR1 secretion [3], was used as a control. The defect in secretion of antimicrobial proteins in the tbf1 mutant correlated with the 1 log higher growth of the bacterial pathogen, Psm ES4326, compared to WT and the complementation lines (FIG. 11D). In response to induction by SA, less than 1 log reduction in Psm ES4326 growth was observed in the tbf1 mutant, compared to the ˜2 log reduction detected in WT plants (FIG. 11E). SA-inducible defenses were restored in the TBF1 complementation line, while the npr1-1 line was completely deficient in establishing resistance.

We also examined elf18-triggered immunity in the tbf1 mutant, because expression profiling data demonstrated that TBF1 was responsible for significant genome-wide transcriptional changes induced by elf18 (FIG. 5C). Leaves were infiltrated with elf18 or another MAMP signal, flg22, and infected with Psm ES4326 4 hours later. WT pre-treated with the MAMP signals showed a 1-log reduction in Psm ES4326 growth, compared to mock-treated samples (FIG. 11F and FIG. 15). The tbf1 mutant, however, completely failed to establish the resistance induced by elf18. This defect was specific to elf18, as flg22-induced resistance was intact in the tbf1 mutant, resembling the trend observed for the growth inhibition phenotype (FIG. 11A and FIG. 15). Since earlier studies demonstrated that flg22 and elf18 induce largely overlapping sets of genes [12], different levels of responsiveness to elf18 and flg22 in tbf1 were unexpected. Our current results are consistent with the genetic data showing that the recognition of elf18, but not flg22, specifically requires the ERQC mechanism [6, 7, 29, 30], and with observations demonstrating that TBF1 controls the induction of these ERQC genes (FIG. 5A). The molecular mechanism underlying the TBF1-requirement has not been determined. TBF1 may affect the biogenesis of EFR, downstream signaling functions, or elf18-triggered secretion of antimicrobial compounds.

A near normal response to elf18 was also observed in the SA-insensitive npr1-1 mutant in the Psm ES4326 infection experiment (FIG. 11F). Although there was an overall increase in Psm ES4326 growth in npr1-1, elf18 could still induce a 1-log reduction in pathogen growth, which was similar to that observed in the WT sample. These results are in agreement with expression profiling data demonstrating that elf18 and SA induce distinct sets of genes (FIG. 5C). Recent studies have also demonstrated that MTI induced by flg22 and elf18, is largely intact in a sid2 mutant, which is deficient in SA synthesis [31]. MTI and SAR, therefore, are two temporally and spatially separate immune responses. MTI occurs locally and immediately upon challenge by a pathogen, while SAR is a systemic response induced after the local response.

Translation of TBF1 is Controlled by uORFs Sensitive to Cellular Metabolic Changes

The genome-wide expression profiling data and the genetic data in the Examples set forth herein demonstrate that TBF1 is a major molecular switch, that upon challenge by a pathogen, turns on multiple defense responses and inhibits primary growth and development (FIGS. 5 and 11). To understand how TBF1 is regulated, we analyzed its expression patterns upon treatment with SA, detecting maximum levels of transcript at 4 hours (FIG. 16A). The SA-dependent induction was abolished in the npr1-1 mutant, demonstrating that NPR1 is required for SA-mediated TBF1 transcription. TBF1 also plays a role in regulating NPR1, as the NPR1 transcript levels were diminished in tbf1 (FIG. 16B), suggesting that a feedback mechanism exists involving these two key immune system regulatory factors.

Analysis of the TBF1 mRNA through the 5′ and 3′ rapid amplification of cDNA ends (RACE) experiments demonstrated that the transcript which encodes TBF1, also comprises two upstream open reading frames (uORFs) (SEQ ID NOS: 108 and 109) (FIG. 16C). The second of these, uORF2 (also known as conserved peptide upstream open reading frame 49, CPUORF49, Arabidopsis gene model AT4G36988) (SEQ ID NO: 103) is well-conserved among TBF1 homologs in other plant species [32]. Translation in eukaryotes normally starts at the first 5′ AUG codon. The presence of two uORFs upstream from (5′ to) the TBF1 start codon, suggests that they may influence the initiation of translation of TBF1. To test this, we made a vector comprising a translational fusion between the 5′ UTR of TBF1 containing both uORFs, the first exon of TBF1, and the GUS reporter gene (abbreviated as uORF1-uORF2-GUS). We also constructed three other vectors which have the start codon mutated (from ATG to CTG) for uORF1 (uorf1-uORF2-GUS), uORF2 (uORF1-uorf2-GUS), and both uORFs (uorf1-uorf2-GUS). To ensure equal levels of transcription in these vectors, we used the constitutive 35S promoter to drive the expression of these reporter genes. The vectors were used in transient-expression experiments carried out in Nicotiana benthamiana leaves, measuring the GUS activity in each sample (FIG. 16D). Using activity of the WT construct as a control, we detected 1.5- and 3.5-fold increases in the GUS activities in uorf1-uORF2-GUS and uORF1-uorf2-GUS samples, respectively. Mutating both uORFs in uorf1-uorf2-GUS resulted in a 3.5-fold elevation in GUS activity over WT, which was similar to the result observed for the uorf2 mutant. These results suggest that both uORFs can function to inhibit TBF1 translation, with uORF2 likely to play a more important role than uORF1 in this matter.

To better understand the regulatory mechanisms involved in TBF1 translation during plant defense events, we measured the GUS activities of the translational fusion vectors in stable transgenic Arabidopsis lines when challenged by Psm ES4326 carrying the avirulent effector, avrRpt2. We found that introduction and recognition of this avirulent bacterial strain, which can induce MTI, ETI, and SAR in plants, caused a rapid increase in the activity of GUS translated from the TBF1 start codon downstream of the uORFs (FIG. 16E). This increase was not observed in the uorf1-uorf2-GUS transgenic lines. These results suggest that the Psm ES4326/avrRpt2 challenge could rapidly alleviate the inhibitory effect of the uORFs on translation of the downstream gene.

To determine whether the endogenous TBF1 was translated in the plant cell upon pathogen challenge, we conducted a polysome profiling experiment shown in FIG. 16F. There was a significant increase in TBF1 transcript 30 minutes after Psm ES4326/avrRpt2 inoculation in the polysomal fractions of the gradient. This rapid association with polysome samples correlates well with the GUS reporter activities observed in the transgenic plants, noted above (FIG. 16E). The association of TBF1 within polysome samples appeared to be transient, as the TBF1 transcript decreased at 1 hour post inoculation. This pathogen-induced derepression of TBF1 translation occurs earlier than the transcription activation of the TBF1 gene (FIG. 16A).

Both uORFs are enriched in aromatic amino acids, particularly in phenylalanine (Phe) (uORF1-27%, and uORF2-19%), as compared to the average frequency of aromatic amino acids reported for species sequenced so far (7.63-7.86%) ([33]; ExPASy proteomics server expasy.org/sprot/relnotes/relstat.html). The enrichment in Phe is evolutionarily-conserved for uORF2 among the TBF1 homologs in other plant species [32]. This suggests that translation of the two uORFs and the downstream TBF1 may be influenced by the cellular availability of Phe for translation, caused by the pathogen challenge. Amino acid starvation has previously been shown to de-repress uORF-mediated translation inhibition on the yeast General Control Nondepressible 4 (GCN4) and the mammalian Activating Transcription Factor 4 (ATF4) genes [34, 35].

To determine if pathogenic infections can trigger changes in amino acid concentrations, we carried out the studies involving the measurement of amino acids in a large number of biological replicates. We could occasionally detect a rapid decrease in the level of Phe that occurred 15 to 45 minutes after Psm ES4326/avrRpt2 inoculation, followed by an increase in the level of Phe that was observed consistently (data not shown). These results suggest that Phe concentrations may change dramatically during early time points after infection, and that it is difficult to measure transient metabolic changes following pathogen challenge using methods that are currently available.

To improve our method of examining whether Phe levels affect the TBF1 translation rate, we used a yeast-based reporter system. Since a Phe-deficient Arabidopsis mutant that has not been identified to date, a yeast chorismate mutase deletion strain, aro7, which is auxotrophic for Phe and tyrosine (Tyr) [36]. A reporter vector was generated by fusing uORF1-uORF2-TBF1_(1st exon) to the coding region of the mouse DHFR (dihydrofolate reductase), which has been engineered to be less stable [37] and resistant to methotrexate (MTX) [38]. DHFR is an enzyme that regulates levels of tetrahydrofolate essential for growth. In the presence of 80 μM of MTX that abolishes the endogenous DHFR enzymatic activity, yeast growth becomes dependent on the recombinant DHFR reporter expression. Since both uORF1-uORF2-TBF1_(1st exon)-DHFR (abbreviated as uORF1-uORF2-DHFR), and the DHFR control, are driven by the endogenous yeast DHFR promoter, growth of these yeast strains reflect the translational rate of DHFR. We cultured the aro7 strain containing either uORF1-uORF2-DHFR or DHFR in presence of MTX. As shown in FIG. 17A, under the Phe-rich conditions (75 mg/L; standard Phe concentration in synthetic yeast growth media), the yeast strain carrying the DHFR displayed a much higher growth rate than the strain carrying the uORF1-uORF2-DHFR, showing the inhibitory effects of the uORFs on DHFR translation. Both strains grew at similar rates under Phe-restricting conditions (15 mg/L), suggesting that low Phe level released the inhibitory effects of uORFs on DHFR translation (FIG. 17A). To ensure that the TBF1_(1st exon)-DHFR fusion protein was not toxic to yeast cells, we grew the strains in the absence of MTX and observed no significant difference in their growth rates (FIG. 18). The uORFs of TBF1 appear to be specifically sensitive to Phe starvation, because aspartic acid starvation caused by addition of 15 mM tobramycin (TOB), a known inhibitor of yeast tRNA^(Asp) aspartylation [39], did not eliminate the difference in the growth rate between the strain carrying uORF1-uORF2-DHFR, and the strain carrying DHFR.

To understand the molecular mechanism by which uORFs control TBF1 translation, we carried out additional experiments. In yeast, amino acid starvation leads to accumulation of uncharged tRNAs, which in turn bind to the HisRS domain of the GCN2 serine/threonine protein kinase, activating it, and causing structural rearrangements within the GCN2 dimer [40, 41]. The activated GCN2 undergoes autophosphorylation, and activating its kinase function involved in phosphorylation of its sole target, eukaryotic initiation factor 2α (eIF2α) [42]. The phosphorylated eIF2α allows initiation of translation, such as GCN4, downstream of uORFs [35]. To determine whether a similar mechanism controls the translation of TBF1 after pathogen infection in plants, we first performed Northern blot analyses to measure the levels of charged and uncharged tRNA after inoculation with Psm ES4326/avrRpt2. As shown in FIG. 17B, a dramatic increase in both charged and uncharged tRNA^(Phe), was measured, appearing as early as 30 minutes after bacterial inoculation, and persisting for 8 hours, compared to only a moderate increase in the level of charged tRNA^(Asp) level under the same conditions. No uncharged tRNA^(Asp) was detected. These results are consistent with observations suggesting that pathogen challenges in plants can lead to dramatic changes in Phe metabolism.

We then investigated whether the pathogen-induced accumulation of uncharged tRNA^(Phe) can lead to phosphorylation of eIF2α, since a functional and stress-inducible GCN2-eIF2α pathway has been found in Arabidopsis [43]. As shown in FIG. 17C, in leaf samples infected with Psm ES4326/avrRpt2, a rapid accumulation of the phosphorylated eIF2α was detected, supporting observations which suggest that it may facilitate re-attachment of the ribosome to the TBF1 translation start codon downstream of uORFs to initiate TBF1 translation.

Taken together, these observations strongly suggest that TBF1 expression is tightly controlled in the plant cell at not only the transcriptional level by NPR1, but also at the translational level through uORFs. Pathogen challenges, which cause a temporary increase in uncharged tRNA^(Phe) accumulation, trigger eIF2α phosphorylation, resulting in de-repression of the translation of TBF1.

DISCUSSION

The presence of TL1 in a wide array of defense-related gene promoters suggests that it plays a critical role as a transcription factor (TF) involved in immune responses in plants [3, 18, 19] (FIGS. 5D and 5E). In these studies, we also demonstrated that the tbf1 mutant plants are impaired in UPR, elf18-induced MTI and SA-mediated SAR, but not in the heat shock response (FIG. 11 and FIG. 13). The evolution of transcriptional factors with novel functions may explain the greater expansion of HSF-like genes in plants compared to other organisms (one each in yeast and Drosophila, and three in vertebrates) [44].

Activation of the immune system consumes a significant amount of metabolic activity. Mutant plants with constitutively-activated defense responses often have stunted growth and retarded development [45]. Our studies demonstrate that TBF1 is a master molecular switch for this growth-to-defense reprogramming that involves activation and repression of nearly 3,000 genes during SAR and MTI. About 46% of these contain at least one copy of the TL1 element in their promoters. TBF1 is involved not only in the control of immune response genes, but also the control of genes relating to primary metabolism, growth, and photosynthesis.

Our analysis revealed seven members of the alpha-expansin gene family in the SA-repressed, TBF1-dependent category. Expansins are cell wall-loosening proteins that mediate pH-dependent extensions of the plant cell wall and growth of the cell [46]. Cell hypertrophy (enlargement) is a common virulence strategy used by bacteria to promote pathogenicity [47-49]. A bacterial effector AvrBs3 from Xanthomonas spp. activates a plant bHLH TF gene, UPA20, which in turn induces multiple alpha-expansin genes [47]. Our study reveals that upon SA signal perception, TBF1 down-regulates expansin that may inhibit this virulence strategy. The presence of TL1 elements in alpha-expansin promoter regions, as shown in Table S1, incorporated as a large table by reference, suggests that they are direct transcriptional targets of TBF1.

The pivotal role that TBF1 plays in the growth-to-defense transition underscores the importance for the need to understand how it regulates other cellular functions and how its expression and activity are regulated. The expression of TBF1 is tightly controlled at both transcriptional and translational levels. Transcription of TBF1 and NPR1 appears to be interdependent, as mutations in either gene affect the transcription of the other gene (FIG. 5A). TBF1 may directly regulate NPR1 expression through the TL1 elements in the NPR1 promoter. Since the NPR1 promoter also contains W-boxes [50], TBF1 may regulate NPR1 indirectly through its transcriptional targets, WRKY TFs (Table S1). NPR1 may also regulate TBF1 expression through either TGA or WRKY TFs, as the TBF1 promoter contains five W-boxes and three TGA binding sites, also known as as-1 elements [51].

The two uORFs upstream of TBF1 ORF link translation of TBF1 with the availability of specific amino acids within the cell. About 10% of all eukaryotic mRNAs contain uORFs, and a high percentage of them encode critical cellular regulators, such as protooncogenes, TFs, receptors, and other proteins involved in immune responses [52]. Expression of these genes is highly-regulated, as their protein products are essential for controlled cell growth and proliferation. TBF1 appears to be a key regulator, as transgenic lines overexpressing TBF1 cDNA under the constitutive 35S promoter were not viable (Pajerowska-Mukhtar and Dong, personal observation).

Pathogen challenges, resulting in increases in uncharged tRNA^(Phe) and the phosphorylation of eIF2α, release the inhibitory effect of uORFs on the translation of TBF1 (FIG. 17D). These results appear to be similar to the regulatory mechanisms described for the well-studied yeast GCN4 and mammalian ATF4 gene products (reviewed in [35]). The yeast GCN4 transcript contains four uORFs in its 5′ regulatory region [53]. Under normal conditions, ribosomes bind to the 5′ cap of GCN4 mRNA and initiate translation at the first uORF. They are unable to reinitiate translation at the start codon of GCN4. During amino-acid starvation, uncharged tRNAs induce phosphorylation of eIF2α mediated by GCN2, which hinders reassembly of the 80S ribosome after translation of uORF1. This allows the 40S ribosomal subunit to continue scanning the mRNA and reinitiate translation at the start codon for GCN4 [53].

While derepression of GCN4 translation can be triggered by starvation of any amino acid, the uORF-mediated regulation of TBF1 in plants appears to be controlled by the metabolic levels of specific amino acids, such as Phe. It is note clear, yet, if an infection by a pathogen causes a transient reduction in the levels of Phe. The rapid increase in the uncharged tRNA^(Phe) after pathogen challenge coincided with the increase of the total tRNA^(Phe) (FIG. 17B). These results suggest that infection by a pathogen affects the availability of Phe required for translation, as aromatic amino acids are known precursors for a large array of plant metabolites, including the growth hormone auxin, the SAR signal SA, cell wall components, and pigments such as anthocyanins [4, 54, 55]. In a manner similar to that observed for GCN4, the accumulation of uncharged tRNA^(Phe) triggers phosphorylation of eIF2α, ribosomal movement through uORFs, and the translation of TBF1. The translational mechanisms involved in the regulation of TBF1 allow the cell to quickly detect pathogen-triggered metabolic changes, and produce sufficient amounts of TBF1 protein to activate cellular and systemic immune responses.

Example 2

Plant disease is a large threat to crop yield and security of the food supply around the world. A variety of approaches have been used to minimize plant disease. NLR (Nucleotide-binding leucine-rich repeat) proteins, PRRs (pattern-recognition receptors), and mutant alleles of host disease-susceptibility genes, for example, have all been used to engineer disease-resistant transgenic plants [85]. Immune responses are energy-consuming processes, adversely affecting plant growth and development. Approaches which stringently control the expression of genes of interest, may minimize the impact of these costly processes. The transcriptional control factor known as TBF1 (TL1 binding factor 1) affects transcriptional reprogramming induced by two important immune signals, elf18 and SA (Salicylic acid) [88]. The level of TBF1 mRNA is rapidly induced by treatment with SA, suggesting that its promoter (TBF1p) is a good candidate for experiments designed to control the transcription of genes of interest in cells infected by a pathogen. The two upstream open reading frames (uORFs) residing in the 5′UTR (5′ untranslated region) of TBF1 mRNA (FIG. 22) appear to suppress the translation of the TBF1 polypeptide. This suppression can be alleviated by infection with a pathogen, suggesting that the uORFs can be used to control translation of genes of interest in pathogen-infected cells. Use of a nucleotide sequence comprising the TBF1 promoter with sequences encoding uORFs may facilitate the control over the level of transcription and translation of disease-related genes and their products. Tissues infected with pathogens have increased accumulation of plant defensive proteins to execute immune response in a spatial and temporal manner. Since the amount of plant defensive proteins are limited in plants, and most of them are specific to particular strains of pathogens, we can exploit target genes from other species, such as toxic or cell death-promoting genes, to locally induce cell death to restrict the growth of a variety of pathogens (exemplified by the list in Table 7).

Recently, the Arabidopsis GCN2 (general control nonrepressed 2; a serine/threonine-protein kinase) protein was shown to directly phosphorylate eIF2α [59]. We previously noted a rapid accumulation of the phosphorylated eIF2α in leaf samples infected with Psm ES4326/avrRpt2 (FIG. 17C). This accumulation temporally correlated with translational de-repression of the uORF1-uORF2-GUS reporter in planta (FIG. 16E). These results suggested that TBF1 might be controlled by a pathogen-mediated induction of the GCN2-eIF2α pathway.

TABLE 7 Characteristics of target genes Synthetic Reference/ Protein name location Major function Source Luciferase Cytosol Positive control for cytosol-synthesized proteins, to facilitate Promega monitoring of both the response of TBF1 promoter and the uORF genetic elements during biotic and abiotic stresses. HA-mBax Cytosol Mammalian apoptosis-promoting protein which causes cell death in [86] plants. NPR1-EGFP Cytosol Master regulator of plant resistance. [84] snc1 Cytosol Constitutively-active resistance protein. [87] snc1-cMyc Cytosol Constitutive active resistance protein with cMyc tag to facilitate the [87] detection of changes in protein accumulation. mGFP5 ER Positive control for polypeptides synthesized in the ER and to monitor [90] the responses of the TBF1 promoter and the uORF genetic elements during biotic and abiotic stresses. Bax-inhibitor 1- ER Polypeptide conferring broad-spectrum resistance to both biotic and [89] HA (Bl-1-HA) abiotic stress.

Materials and Methods

A variety of primers used in the construction of various plasmid vectors comprising genetic elements including promoters and sequences encoding polypeptides of interest are listed in Table 8. Key features of plasmid vectors used in this study are listed in Table 9.

TABLE 8 Primers used in plasmid construction SEQ ID Name Sequence Length Description Reference NO: P1 cggCTGCAGgtcaacatggtggagcacga 29 PstI-35S 5′ This study 115 P2 cggTCTAGAccggcctctccaaatgaaatgaac 33 XbaI-35S-3′ This study 116 p3 cggGGTACCgatcgttcaaacatttggcaata 32 KpnI-NOS 5′ This study 117 P4 cggGAATTCcccgatctagtaacatagatg 30 EcoRI-NOS 3′ This study 118 P5 cggGGTACCttcgacgacaagaccgggcccacaagtt 55 KpnI-Gateway 5′ This study 119 tgtacaaaaaagctgaac P6 ggaaattcgagcggctcgagtgaggagaagagccggg 61 Gateway 3′ This study 120 cccctaccactttgtacaagaaag P7 cggCTTAAGaaactttattgccaaatgtttgaacgat 58 AflII-Gateway 3′ This study 121 cggggaaattcgagcggctcg P8 cggGGTACCctccggcgaactttttttattt 31 KpnI-TBF1 5′UTR This study 122 3′ P9 aattTCTAGAaacagcatccg 21 XbaI-TBF1 5′UTR This study 123 with native uORFs5′ P10 cggTCTAGAaacagcatccgtttttataatttaattt 55 XbaI-TBF1 5′UTR This study 124 tcttacaaaggtaggacc with mutant uORFs 5′ P11 cggAAGCTTcgacgactagtttacagagaa 30 HindIII-TBF1 This study 125 promoter 5′ P12 cggGGCGCGCCctagaaattctcagaaacatcttttc 40 AscI-TBF1 This study 126 ttc promoter 3′ P13 cggGGCGCGCCttcttacaaaggtaggaccaac 33 AscI-TBF1 5′UTR This study 127 5′ P14 cggAAGCTTtacagagaatttggaccgtc 29 HindIII-TBF1 This study 128 promoter 5′ P15 cggACTAGTaattctcagaaacatcttttcttc 33 SpeI-TBF1 This study 129 promoter 3′ LIC1 tcgacgacaagacc 14 Gateway LIC This study 130 adapter sequence 1 LIC2 tgaggagaagagcc 14 Gateway LIC This study 131 adapter sequence 2

The 35S promoter, with duplicated enhancer elements, was amplified from pRNAi-LIC (GenBank: GQ870263.1) using primers P1 (SEQ ID NO: 115)/P2 (SEQ ID NO: 116) and was flanked with PstI and XbaI sites. The NOS terminator was amplified from pRNAi-LIC (GenBank: GQ870263.1) using primers P3 (SEQ ID NO: 117)/P4 (SEQ ID NO: 118) to produce a DNA sequence which is flanked with KpnI and EcoRI sites. The Gateway cassette with LIC adapter sequences

(SEQ ID NO: 130) LIC1: tcgacgacaagacc (SEQ ID NO: 131) LIC2: tgaggagaagagcc were amplified using primers P5 (SED ID NO: 119)/P6 (SED ID NO: 120)/P7 (SED ID NO: 121) (the PCR fragment by P5/P6 was used as template for P5/P7) from pDEST375 (GenBank: KC614689.1) and was flanked with KpnI and AflII sites. The NOS terminator, 35S promoter, and Gateway cassette were sequentially ligated into pCAMBIA1300 (GenBank: AF234296.1) via KpnI/EcoRI, PstI/XbaI and KpnI/AflII. The resulting plasmid (designated pGXO (SEQ ID NO: 136) was used as an intermediate plasmid.

The 5′UTR of TBF1 with native or mutant uORFs were amplified with P8 (SED ID NO: 122)/P9 (SED ID NO: 123) and P8 (SED ID NO: 122)/P10 (SED ID NO: 124) from uORF1-uORF2-GUS and uorf1-uorf2-GUS plasmids as previously published [88] respectively, and were cloned into the intermediate plasmid via XbaI/KpnI. The resulting plastmids were designated as pGX180 (35S-uORF-Gateway-NOS) (SEQ ID NO:135) and pGX179 (35S-uorf-Gateway-NOS) (SEQ ID NO: 134), respectively.

The TBF1 promoter was amplified from Arabidopsis Genomic DNA using primers P11 (SED ID NO: 125)/P12 (SED ID NO: 126) and was flanked with HindIII/AscI. The TBF1 5UTR was amplified from pGX180 using primers P8 (SED ID NO: 123)/P13 (SED ID NO: 127) and was flanked with AscI/KpnI. The TBF1 promoter (P11 (SED ID NO: 125)/P12 (SED ID NO: 126)) and TBF1 5UTR (P8 (SED ID NO: 122)/P13 (SED ID NO: 127)) were digested with AscI and ligated together. The ligation product was used as template for amplifying the TBF1 promoter-5′UTR fusion PCR product with primer pair P11 (SED ID NO: 125)/P8 (SED ID NO: 122), which produced a DNA fragment that was flanked with HindIII/KpnI sites. The 35S promoter-uORF region on pGX179 was also replaced by the TBF1 promoter-5′UTR, and the resulting plasmid was designated as pGX1 (TBF1p-uORF-Gateway-NOS) (SEQ ID NO: 132).

The TBF1 promoter was amplified from Arabidopsis genomic DNA using primers P14 (SEQ ID NO: 128)/P15 (SEQ ID NO: 129) and was flanked with HindIII/SpeI and was ligated into pGX179 (SEQ ID NO: 134) which was cut with HindIII/XbaI (generating SpeI-compatible sticky ends). The resulting plasmid was designated pGX181 (TBF1p-uorf-Gateway-NOS) (SEQ ID NO: 133).

TABLE 9 Plasmids SEQ Length Reference/ ID Designation Markers Description (nt/aa) Source NO pGX1 Kan^(R), pGX1 (TBF1p-uORF-Gateway-NOS) 14478 This study 132 Hygro^(R) Gateway plant expression vector carrying a Gateway cassette cloned downstream of uORF1 and uORF2. Target genes can be cloned via Ligation independent cloning method [91] to replace Gateway cassette. The target gene with uORFs on its 5′ is driven by TBF1 promoter; vector confers kanamycin resistance in E. coli and hygromycin resistance in transgenic plants. pGX181 Kan^(R), pGX181 (TBF1p-uorf-Gateway-NOS) 14488 This study 133 Hygro^(R) A-to-C point mutation was introduced into the start codons (ATG) of uORF1 and uORF2, designated as uorfl-uorf2. Gateway plant expression vector carrying a Gateway cassette cloned downstream of uorfl and uorf2. Target genes can be cloned via Ligation independent cloning method [91 ]to replace Gateway cassette. The target gene with uorfs on its 5' is driven by TBF1 promoter; vector confers kanamycin resistance in E. coli and hygromycin resistance in transgenic plants. pGX179 Kan^(R), pGX179 (35S-uORF-Gateway-NOS) 12194 This study 134 Hygro^(R) A-to-C point mutation was introduced into the start codons (ATG) of uORF1 and uORF2, designated as uorfl-uorf2. Gateway plant expression vector carrying a Gateway cassette cloned downstream of uorfl and uorf2. Target genes can be cloned via Ligation independent cloning method [91 ]to replace Gateway cassette. Expression of the target gene with uorfs on its 5' end is driven by 35S promoter. The vector confers kanamycin resistance in E. coli and hygromycin resistance in transgenic plants. pGX180 Kan^(R), pGX180 (35S-uorf-Gateway-NOS) 12187 This study 135 Hygro^(R) Gateway plant expression vector carrying a Gateway cassette cloned downstream of uORF1 and uORF2. Target genes can be cloned via Ligation independent cloning method [91 ] to replace Gateway cassette. Expression of the target gene with uORFs on its 5' end is driven by 35S promoter. The vector confers kanamycin resistance in E. coli and hygromycin resistance in transgenic plants. pGX0 Kan^(R), pGX0 Intermediate plasmid 11726 This study 136 Hygro^(R) Gateway plant expression vector carrying a Gateway cassette cloned downstream of 35S promoter. The vector confers kanamycin resistance in E. coli and hygromycin resistance in transgenic plants. This intermediate vector was used in the construction of pGX1, pGX181, pGX179, and pGX180. Luciferase Positive control for cytosol-synthesized 1653 Promega 137 proteins, to facilitate monitoring of both the response of TBF1 promoter and the uORF genetic elements during biotic and abiotic stresses. Luciferase Luciferase polypeptide 550 aa Promega 138 NPR1-EGFP Arabidopsis NPR1 gene that controls systemic 2532 This study 139 acquired resistance encodes a novel protein PCR amplify containing ankyrin repeats [84]. from Arabidopsis cDNA and PCR fuse with EGFP NPR1-EGFP NPR1-EGFP fusion polypeptide 843 aa This study 140 HA-mBax Mammalian apoptosis-promoting protein 780 This study 141 which causes cell death in plants [86]. PCR amplify from Mouse cDNA and PCR fuse with HA tag, HA-mBax HA-mBax fusion polypeptide 259 aa This study 142 mGFP5 ER localized GFP used as positive control for 792 [90] 143 polypeptides synthesized in the ER and to monitor the responses of the TBF1 promoter and the uORF genetic elements during biotic and abiotic stresses [90]. mGFP5 mGFP5 polypeptide 263 aa [90] 144 BI-1-HA Polypeptide conferring broad-spectrum 936 [89] 145 resistance to both biotic and abiotic stress [89]. Amplify PCR fragment from Arabidopsis cDNA and fuse with HA fragment. BI-1-HA BI-1-HA fusion polypeptide 311 aa [89] 146 snc1 Genomic fragment of Arabidopsis snc1 mutant 4950 This study 147 plant encoding constitutively-active form of PCR amplify SNC1 resistance protein (suppressor of NPR1) from genomic [87]having 6 exons and 5 introns. DNA of snc1 mutant plant. snc1-cMyc Nucleotide sequence encoding constitutively- 5244 This study 148 active resistance protein [87]fused via linker sequence to cMyc on its C terminal end. Snc1 portion is encoded by nucleotide sequence having 6 exons and 5 introns, where the snc1- related exons encode a polypeptide fused via linker to a cMyc polypeptide. Linker portion of snc1-linker-cMyc fusion 11 aa 149 polypeptide. cMyc portion of snc1-linker-cMyc fusion 78 aa 150 polypeptide

Results

Four different versatile vectors were generated using the Gateway system and ligation-independent cloning strategy as illustrated in FIG. 23 [91]. A variety of genes were cloned into the four expression cassettes using a ligation independent cloning method [91].

Use of the uORF elements to control expression of luciferase (cytosol-synthesized protein) and mGFP5 (ER-synthesized protein) demonstrated that TBF1 uORF can suppress both cytosol- and ER-synthesized proteins (FIG. 24), which enables the secreted proteins and membrane proteins as the potential targets.

In this example, we transformed the uORF1-uORF2-GUS construct into the Arabidopsis gcn2 knock-out mutant and the corresponding Landsberg erecta (Ler) wild-type plants. To test whether GCN2 controls TBF1 translation via uORFs, we also created an additional set of transgenic lines in the Ler background that express a derivative construct in which the start codons for both uORFs (uorf1-uorf2-GUS) were mutated (from ATG to CTG). GUS activities of these translational fusions were quantified in T₃ stable transgenic Arabidopsis lines in response to Psm ES4326/avrRpt2.

We observed a rapid increase in the GUS activities only in the wild-type Ler plants expressing uORF1-uORF2-GUS (FIG. 21). This increase was not observed in the gcn2 mutant carrying uORF1-uORF2-GUS or wild-type expressing uorf1-uorf2-GUS. These data demonstrate that TBF1 translation is mediated by the GCN2/eIF2α pathway upon pathogen infection. Since GCN2 kinase homologs have been found across kingdoms from yeast to animals to plants, the uORF-mediated translation regulatory mechanism discovered for the Arabidopsis TBF1 mRNA is likely to function in a heterologous genetic background. Based on this observation, we replaced the GUS reporter gene with several functional “cargo” genes and transformed them into Arabidopsis. Transgenic plants will be characterized with regard to levels of disease resistance as well as plant fitness.

Transgenic Arabidopsis plants generated by the floral dip method [92] are being assessed for fitness and disease resistance.

While the preferred embodiments of the invention have been illustrated and described in detail, it will be appreciated by those skilled in the art that various changes can be made therein without departing from the spirit and scope of the invention. Accordingly, the particular arrangements disclosed are meant to be illustrative only and not limiting as to the scope of the invention, which is to be given the full breadth of the appended claims and any equivalent thereof.

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All references, patents, or applications cited herein are incorporated by reference in their entirety, as if written herein.

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What is claimed is:
 1. A vector comprising a nucleic acid comprising in a 5′ to 3′ direction a regulatory region to modulate the expression of one or more polypeptides in a cell, wherein said regulatory region comprises a promoter, functional in said cell, operably-linked in a 5′ to 3′ direction to at least one upstream open reading frame (uORF); one or more heterologous sequences selected from the group consisting of a synthetic polylinker, a sequence recognized by a restriction enzyme designated as a cloning site (CS), and a ligation independent cloning sequence (LIC); and one or more heterologous coding sequences comprising a downstream open reading frame (dORF) encoding the one or more polypeptides; wherein said uORF encodes a polypeptide selected from the group consisting of: (i) a polypeptide designated as uORF1 of SEQ ID NO: 102; (ii) a variant of the polypeptide designated as uORF1 of SEQ ID NO: 102 that contains one or two substitutions , and in which the translation control function of uORF1 is conserved; (iii) a polypeptide designated as uORF2 of SEQ ID NO: 103; and (iv) a variant of the polypeptide designated as uORF2 of SEQ ID NO: 103 that contains one or two substitutions, and in which the translation control function of uORF2 is conserved.
 2. The vector of claim 1, wherein said nucleic acid comprises both a sequence encoding a polypeptide selected from the group consisting of the polypeptide designated as uORF1 of SEQ ID NO: 102 and the variant of uORF1 of SEQ ID NO: 102 and a sequence encoding a polypeptide selected from the group consisting of the polypeptide designated as uORF2 of SEQ ID NO: 102, and the variant of uORF2 of SEQ ID NO:
 103. 3. The vector of claim 1, wherein at least one dORF encodes a polypeptide selected from the group consisting of: (i) a polypeptide that is a transcription factor; (ii) a reporter polypeptide; (iii) a polypeptide that confers resistance to drugs or agrichemicals; (iv) a polypeptide involved in resistance of plants to viral, bacterial, fungal pathogens, oomycete pathogens, phytoplasmas, and nematodes; and (v) a polypeptide involved in the growth or development of plants.
 4. The vector of claim 3, wherein said polypeptide comprises a TL1-Binding Transciption Factor 1 (TBF1) polypeptide.
 5. The vector of claim 3, wherein said polypeptide is (ii) a reporter polypeptide or (iii) a polypeptide that confers resistance to drugs or agrichemicals that is selected from the group consisting of: β-galactosidase (β-gal), β-glucuronidase (β-gluc), chlorophenicol acetyltransferase (CAT), Renilla-luciferase (ruc), Photinus luciferase (luc), secreted alkaline phosphatase (SAP), and green fluorescent protein (GFP).
 6. The vector of claim 1, wherein said promoter is selected from the group consisting of: (a) a plant promoter; (b) a plant virus promoter; (c) a promoter from a non-viral plant pathogen; (d) a mammalian cell promoter; and (e) a mammalian virus promoter.
 7. The vector of claim 6, wherein said promoter is a plant promoter.
 8. The vector of claim 7, wherein said plant promoter comprises a TBF1 promoter.
 9. The vector of claim 7, wherein said plant promoter is a nucleotide sequence comprising a binding site for the TBF1 polypeptide in which one or more promoter functions are preserved.
 10. The vector of claim 7, wherein said plant promoter is a nucleotide sequence comprising a functionally-active promoter from Arabidopsis.
 11. The vector of claim 7, wherein said plant promoter is inducible upon challenge by a plant pathogen or a chemical inducer.
 12. The vector of claim 11, wherein said inducer is selected from the group consisting of salicylic acid, Jasmonic acid, methyl ester of jasmonic acid, abscisic acid, ethylene, AgN03, cycloheximide, mannitol, NaCl, flg22, elf18, and LPS.
 13. A modified cell comprising the vector of claim
 1. 14. A modified cell comprising a nucleic acid comprising in a 5′ to 3′ direction a regulatory region to modulate the expression of one or more polypeptides in a cell, wherein said regulatory region comprises a promoter, functional in said cell, operably-linked in a 5′ to 3′ direction to at least one upstream open reading frame (uORF); one or more heterologous sequences selected from the group consisting of a synthetic polylinker, a sequence recognized by a restriction enzyme designated as a cloning site (CS), and a ligation independent cloning sequence (LIC); and one or more heterologous coding sequences comprising a downstream open reading frame (dORF) encoding one or more polypeptides; wherein said uORF encodes a polypeptide selected from the group consisting of: (i) a polypeptide designated as uORF1 of SEQ ID NO: 102; (ii) a variant of the polypeptide designated as uORF1 of SEQ ID NO: 102 that contains one or two substitutions, and in which the translation control function of uORF1 is conserved; (iii) a polypeptide designated as uORF2 of SEQ ID NO: 103; and (iv) a variant of the polypeptide designated as uORF2 of SEQ ID NO: 103 that contains one or two substitutions, and in which the translation control function of uORF2 is conserved.
 15. A plant propagation material comprising one or more modified cells of claim
 14. 16. A plant comprising one or more modified cells of claim
 14. 17. A transgenic plant comprising comprising a nucleic acid comprising in a 5′ to 3′ direction a regulatory region to modulate the expression of one or more polypeptides in a cell, wherein said regulatory region comprises a promoter, functional in said cell, operably-linked in a 5′ to 3′ direction to at least one upstream open reading frame (uORF); one or more heterologous sequences selected from the group consisting of a synthetic polylinker, a sequence recognized by a restriction enzyme designated as a cloning site (CS), and a ligation independent cloning sequence (LIC); and one or more heterologous coding sequences comprising a downstream open reading frame (dORF) encoding one or more polypeptides; wherein said uORF encodes a polypeptide selected from the group consisting of: (i) a polypeptide designated as uORF1 of SEQ ID NO: 102; (ii) a variant of the polypeptide designated as uORF1 of SEQ ID NO: 102 that contains one or two substitutions, and in which the translation control function of uORF1 is conserved; (iii) a polypeptide designated as uORF2 of SEQ ID NO: 103; and (iv) a variant of the polypeptide designated as uORF2 of SEQ ID NO: 103 that contains one or two substitutions, and in which the translation control function of uORF2 is conserved.
 18. A method of modulating the expression of one or more polypeptides in a cell, the method comprising the steps of: (a) introducing the vector of claim 1 into a cell; and (b) expressing the one or more upstream ORFs and the one or more downstream ORFs encoding one or more polypeptides for a period sufficient to modulate the amount or level of activity of at least one of the one or more polypeptides within the cell or in a cell culture medium obtained from said cell.
 19. The vector of claim 1, wherein the vector is selected from the group consisting of: SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, and SEQ ID NO:
 135. 20. The vector of claim 3, wherein said polypeptide comprises a NPR1 polypeptide. 